Maltosyl-isomaltooligosaccharides

ABSTRACT

The application describes compositions that include maltosyl-isomalto-oligosaccharides with a desirable mass average molecular weight distribution. In some cases, the compositions can contain at least 3% mannitol.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority to the filing date of U.S.Provisional Application Ser. No. 62/280,026, filed Jan. 18, 2016, thecontents of which are specifically incorporated herein by reference intheir entity.

BACKGROUND

Dietary supplements are either food or food constituents thatpurportedly provide medical or health benefits such as prevention ofdisease (Stephen, D. F. L., Trends in Food Sci. Tech, 1995, 6:59-61).The term typically includes the following representative classes:probiotics, prebiotics, dietary fiber, omega-3 fatty acids andantioxidants (Pandey, M. et al., Asian J. Pharm. Clin. Res., 2010,3:11-15). Due to increasing numbers of health conscious consumers inAsia, the United States, and Europe, the dietary supplement market,specifically in the area of oligosaccharides and prebiotics, hasdemonstrated significant growth over the last three decades (Goffin, D.et al., Crit. Rev. Food. Sci. Nutr., 2011, 51:394-409; Roberfroid, M.B., Br. J. Nutr., 2002, 88 Suppl 2:S133-8). New, improved products aswell as new, economical methods for their production are in demand.

Prebiotics are materials, or mixtures thereof, that contain eitherphysical (e.g. dietary fiber) or chemical (e.g. butyrate) entities thatcan survive transit through the upper gastrointestinal tract, and canarrive intact in the colon to promote the growth of selected beneficial(probiotic) flora (Chung, C. H., et al., Poult. Sci., 2004, 83:1302-6).In some cases, prebiotics can exert some beneficial effect directly onintestinal epithelial cells such as improving uptake of nutritivecalories, vitamins, minerals, and other beneficial materials. Becausemany prebiotics can overcome the resistance of the digestive barrier tofacilitate the proliferation and/or activity of desired populations ofbacteria in situ (Gibson G. R. et al., J. Nutr., 1995, 125:1401-12; VanLoo, J. et al., Br. J. Nutr., 1999, 81:121-32), research and developmentin this area has boomed. Additionally, some prebiotics are naturallypresent in the food supply, especially in fermented foods, and aregenerally compatible with most food formulations (Macfarlane, S. et al.,Aliment Pharmacol. Ther, 2006, 24:701-14; Manning, T. S. et al., BestPract. Res. Clin. Gastroenterol, 2004, 18:287-98).

Some resistant glucooligosaccharide prebiotic agents may be commerciallyavailable, but some types of glucooligosaccharides are better prebioticagents than others.

Day & Chung have produced glucooligosaccharides, includingmaltosyl-isomaltooligosaccharides (MIMOs) via fermentation (U.S. Pat.No. 7,291,607). However, Day & Chung do not disclose the compositions orthe methods of production described in this application.

SUMMARY

Compositions described herein contain resistant oligosaccharides, wheremaltose is a constituent part. Referred to asmaltosyl-isomaltooligosaccharides (MIMOs), these compositions promotethe growth of beneficial intestinal bacteria, including Lactobacillusand/or Bifidobacterium spp., over non-beneficial bacteria in theintestine.

The application describes compositions and methods of production thereofthat include, for example, maltosyl-isomaltooligosaccharides with a massaverage molecular weight distribution of about 300 to 1500 daltons, orabout 400 to 1200 daltons, or about 640 to 1000 daltons. In some cases,the mass average molecular weight distribution of themaltosyl-isomaltooligosaccharides is about 730 to 900 daltons. Themaltosyl-isomaltooligosaccharides in the compositions generally containmore α-(1-6) glucosyl linkages than α-(1,2), α-(1,3), or α-(1,4) glucoselinkages. For example, the maltosyl-isomaltooligosaccharides can have atleast 50% α-(1,6) glucosyl linkages, or at least 52% α-(1,6) glucosyllinkages, or at least 55% α-(1,6) glucosyl linkages, or at least 60%α-(1,6) glucosyl linkages, or at least 65% α-(1,6) glucosyl linkages, orat least 70% α-(1,6) glucosyl linkages, or at least 75% α-(1,6) glucosyllinkages, or at least 80% α-(1,6) glucosyl linkages, or at least 83%α-(1,6) glucosyl linkages, or at least 85% α-(1,6) glucosyl linkages, orat least 87% α-(1,6) glucosyl linkages, or at least 88% α-(1,6) glucosyllinkages, or at least 89% α-(1,6) glucosyl linkages, or at least 90%α-(1,6) glucosyl linkages. Some of the maltosyl-isomaltooligosaccharidesin the composition can optionally have one or two α-(1,4) glucosyllinkages, or one or two α-(1,2) glucosyl linkages, or one or two α-(1,3)glucosyl linkages. Hence, the maltosyl-isomaltooligosaccharides aregenerally linear α-(1,6) glucooligosaccharides, terminated with maltoseat the reducing end. The sugar residues at the reducing ends of themaltosyl-isomaltooligosaccharides are typically connected to the rest ofthe MIMO molecule via an α-(1,4) linkage.

While in some cases the compositions can have little or no mannitol, thecompositions typically have some mannitol in them. For example, thecompositions can have more than 3%/brix mannitol, or more than 4% %/brixmannitol, or more than 5% %/brix mannitol, for example, as detected byrefractive HPAEC-PAD or HPLC-RID. Generally, the amount of mannitol inthe compositions is less than 30%/brix mannitol, or less than 20%/brixmannitol, or less than 15%/brix mannitol or less than 12%/brix mannitol,or less than 10%/brix mannitol, or less than 9%/brix mannitol, or lessthan 8%/brix mannitol, for example, as detected by HPAEC-PAD orHPLC-RID.

The maltosyl-isomaltooligosaccharides in the compositions generally haveno more than about 17 glucosyl units, or no more than about 16 glucosylunits, or no more than about 15 glucosyl units, or no more than about 14glucosyl units, or no more than about 13 glucosyl units, for example, asdetected by HPAEC-PAD or HPLC-RID.

The compositions generally have less than 2%/brix isomaltose, or lessthan 1%/brix isomaltose, or less than 0.5%/brix isomaltose, or less than0.2%/brix isomaltose, or less than 0.1%/brix isomaltose as detected byHPAEC-PAD or HPLC-RID. In some cases, the compositions have noisomaltose, or levels below the detection limit (for example, asdetected by HPAEC-PAD or HPLC-RID).

The compositions also generally have less than 5%/brix free glucose, orless than 4%/brix free glucose, or less than 3%/brix free glucose, orless than 2%/brix free glucose, or less than 1%/brix free glucose, forexample, as detected by HPAEC-PAD or HPLC-RID.

The compositions also typically have less than 5%/brix sucrose, or lessthan 4%/brix sucrose, or less than 3%/brix sucrose, or less than 2%/brixsucrose, for example, as detected by HPAEC-PAD or HPLC-RID.

The compositions also generally have less than 4%/brix fructose, or lessthan 3%/brix fructose, or less than 2%/brix fructose, or less than1%/brix fructose, or less than 0.5%/brix fructose, or less than0.25%/brix fructose, for example, as detected by HPAEC-PAD or HPLC-RID.

The compositions typically contain small or non-detectable quantities oforganic acids such as lactic acid, acetic acid or formic acid. Forexample, the compositions can have less than 16%/brix lactic acid,acetic acid and formic acid; less than 3%/brix lactic acid, acetic acidand formic acid; less than 2%/brix lactic acid, acetic acid and formicacid; or less than 1%/brix lactic acid, acetic acid, and formic acid; orless than 0.5%/brix lactic acid, acetic acid, and formic acid; or lessthan 0.2%/brix lactic acid, acetic acid, and formic acid; or less than0.1%/brix lactic acid, acetic acid, and formic acid, for example, asdetected by HPAEC-PAD or HPLC-RID. In some cases, the compositions canhave no organic acids such as lactic acid, acetic acid or formic acid,as measured by HPAEC-PAD or HPLC-RID.

The compositions also have low amounts of free maltose. For example, thecompositions typically have less than 8%/brix maltose, or less than7%/brix maltose, or less than 6%/brix maltose, or less than 5%/brixmaltose, for example, as detected by HPAEC-PAD or HPLC-RID.

The compositions can be administered or ingested by animals, includinghumans, domesticated animals, zoo animals, and wild animals. Hence,methods are described herein that involve administering any of thecompositions described herein to an animal. Such administration oringestion can have beneficial effects for the animal. For example,animals that can benefit from such administration or ingestion may havediseases or conditions such as cancer, pre-cancerous condition(s),cancerous propensities, diabetes (e.g., type 2 diabetes, or type 1diabetes), autoimmune disease(s), acid reflux, bacterial infections(e.g., in the mouth, sinuses, and/or gastrointestinal tract), vitamindeficiencies, mood disorder(s), degraded mucosal lining(s), ulcerativecolitis, digestive irregularities (e.g., Irritable Bowel Syndrome, acidreflux, constipation, or a combination thereof), inflammatory boweldisease, ulcerative colitis, Crohn's disease, gastroesophageal refluxdisease (GERD), infectious enteritis, antibiotic-associated diarrhea,diarrhea, colitis, colon polyps, familial polyposis syndrome, Gardner'sSyndrome, Helicobacter pylori infections, intestinal cancers, orcombinations thereof. The compositions can foster the growth andactivity of certain types of bacteria (e.g., L. lactis strains), whichleads to the production of various types of bacteriocins (e.g., nisins)by those bacteria. Such bacteriocins (e.g., nisins) can act asanti-cancer agents and/or as anti-microbial agents, that can reduce thesymptoms, or treat, the diseases or conditions.

The application also describes methods of making the compositions. Forexample, the compositions can be generated by a method that involves thefollowing:

-   -   (a) contacting Leuconostoc citreum ATCC 13146 (NRRL B-742)        bacterial cells with a aqueous culture medium comprising a ratio        of sucrose to maltose ranging from 2.0 to about 4.5 to form a        fermentation mixture;    -   (b) fermenting the fermentation mixture at a pH between 4 and 8;    -   (c) removing the bacterial cells to generate a cell-free liquor;    -   (d) polishing the cell-free liquor by removal of insoluble        impurities;        -   decolorization (e.g., using activated charcoal, activated            carbon, a weak base anion resin, or a combination thereof),            de-ashing (e.g., using a strong acid cation resin to remove            metal ions, or using a two-step process using a strong acid            followed by a weak base); removing protein (e.g., by            heating, evaporating the aqueous culture medium, and            centrifugation or filtration, or by using a weak base anion            resin); removing organic acids (e.g., utilizing a weak base            anion resin, liquid chromatography using a chromatographic            grade gel-type strong acid cation exchange resin in calcium            form (SAC-Ca⁺⁺); or any combination thereof, to generate a            polished product;    -   e) washing insoluble impurities, decolorization agents,        de-aching agents, evaporating mechanisms (e.g., a wiped film        evaporator), centrifugation pellets, filters, ration, weak base        anion resins, chromatographic resins, chromatographic grade        gel-type strong acid cation exchange resins, or any combination        thereof to generate one or more washes; and combining one or        more washes together, or with the cell-free liquor; or with the        polished product;    -   wherein the final composition comprises        maltosyl-isomaltooligosaccharides with a mass average molecular        weight distribution of about 640 to 1000 daltons and at least 3%        mannitol.

In some cases, the ratio of sucrose to maltose in step (a) ranges fromabout 2.0 to about 4.5, or about 2.1 to about 4.0, or about 2.2 to about3.5, or about 2.3 to about 3.0, or about 2.5 to about 3.0, or about 2.5to about 2.9, or about 2.5 to about 2.8, or about 2.75. These ratios ofsucrose to maltose are at the time of inoculation.

Because the pH of the biosynthetic methods used to make themaltosyl-isomaltooligosaccharide compositions is controlled at theoptimum for dextransucrase enzyme(s), the resulting composition is not aproduct of nature. In nature, bacteria will in the course of metabolicprocesses, create organic acids. As this occurs, the pH will drop to3.5-4.2. The ultimate pH in a natural product is thus significantly lessthan the optimum range of dextransucrase enzymes (e.g. pH 5.6, see FIG.7) which will limit both the yield and the mass average molecular weightdistribution of MIMOs so made. Hence a product made in nature willtypically have a degree of polymerization (DP) range between 3 and 6,weighted heavily towards DP 3. In comparison, the DP of the compositionsdescribed herein can be higher. For example, most of the MIMOoligosaccharides in the compositions provided herein have a DP of 4 ormore, or even a DP of 5 or more. An example of a MIMO compositionprepared as described herein is shown in FIG. 39.

For example, at least 40%, or at least 50%, or at least 55%, or at least57%, or at least 60%, or at least 62%, or at least 65% of the MIMOs inthe compositions describe here have a DP of 5 or more.

Furthermore, the compositions produced as described herein aresignificantly free of organic acids, but those organic acids will bepresent in a natural fermented product. Also, under equivalentconditions in terms of the quantity of sucrose present, the naturalproduct will contain significantly more mannitol (e.g., 33%/brix) thanthe composition described herein (typically 6-8%/brix).

DESCRIPTION OF THE FIGURES

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates the metabolism of Leuconostoc spp. (see, Dols, et al.1997).

FIG. 2 shows High-Performance Anion-Exchange Chromatography with PulsedAmperometric Detection (HPAEC-PAD) of various oligosaccharidepreparations: (1) a final fermentation broth generated using theISOThrive process with NRRL B-1299; (2) a commercial MIMO formulationmade using an immobilized dextransucrase enzyme from NRRL B-1299; and(3) a typical product made using a process described in U.S. patentapplication Ser. No. 14/833,094, filed Aug. 22, 2015 (incorporatedherein by reference in its entirety) using NRRL B-742. Note thatequivalent components a-j are observed, which correspond to a. sucrose,b. maltose, and c-j corresponding to MIMO with (degree of polymerization(DP) 3-9, respectively. Subscript C1 denotes MIMO with (1) α-(1,6)linear chains; (2) α-(1,2) branched chains; and, (3) α-(1,3) branchedchains.

FIG. 3 shows HPAEC-PAD of (1) fermentation medium made to containISOThrive™ MIMO corresponding to “A2”-type material. (2), (3), and (4)correspond to the same medium after fermentation with L. casei NRRLB-1922 for 3, 5, and 7 days, respectively. Labeled components are A:D-mannitol; B: L-arabinose (internal standard); C: D-glucose; D:sucrose; E: maltose; F: MIMO-DP3 (e.g., panose), and G-K: MIMO DP 4-8.Note that consumption of MIMO occurs via cleavage of maltose anddepolymerization of the α-(1,6) glucan backbone chain.

FIG. 4 shows HPAEC-PAD of (1) Fermentation medium made to contain acommercial MIMO product manufactured using immobilized dextransucraseisolated from NRRL B-1299. (2) and (3) correspond to the same mediumafter fermentation with L. casei NRRL B-1922 for 3 and 6 days,respectively. Labeled components are A: L-arabinose (internal standard);B: D-glucose; C: D-fructose; D: maltose; where E, F, G, and H correspondto MIMO with linear α-(1,6) backbones ranging in DP from 3 to 6, and E2,F2, G2, and H2 correspond to MIMO with linear α-(1,6) backbones andeither α-(1,2) or α-(1,3) glucosyl branches ranging in DP from 3 to 6.Note preferential consumption of the linear α-(1,6) molecules over thebranched moieties. Note also the same product distribution/mechanism asindicated in FIG. 3.

FIG. 5 HPAEC-PAD of (1) MIMO produced in a spontaneously fermentedsourdough starter culture from hard red wheat (confirmed P. pentosaceusvia sequencing of 16SrRNA); (2) Liquor separated from kimchi (found tocontain several Leuconostoc spp., primarily L. gasicomitatum, viasequencing of 16SrRNA) with sucrose and maltose added at a ratio of 3:1;and (3) A sample of ISOThrive™ MIMO produced via fermentation of sucroseand maltose with NRRL B-742. Labeled components are A: sucrose; B.raffinose (internal standard), C: maltose, where D, E, F, G, H, I, L, M,and N correspond to MIMO with α-(1,6) linear backbones ranging in DPfrom 3 (panose) to 10.

FIG. 6 shows the effect the sucrose:maltose (S/M, w/w) ratio at the timeof inoculation has on the mass average molecular weight distribution(MWD) at a fixed pH of 5.5. As S/M increases, so does the molecularweight distribution.

FIG. 7 shows the pH optimum for dextransucrase produced by Leuconostocmesenteroides NRRL B-512F either without (white squares) or with (blacksquares) stabilization via added dextran T-70 (Monchois, et al. 1998).The mass average molecular weight distribution (MWD) increases as theoptimum is approached.

FIG. 8 shows the heating/cooling curve employed during a typicalsterilization in place (SIP) cycle run on a New Brunswick BioFlo 410fermenter.

FIG. 9 shows the behavior of fermentation pH during the course of a 14 Lfermentation with Leuconostoc citreum ATCC 13146, the designation NRRLB-742. Note onset of pH control with NaOH (40% w/w) to maintain a pH of5.50 at approximately 6 hours into log-growth phase.

FIG. 10 shows the progress of fermentation via optical density (OD)through log-phase growth of a 14 L fermentation with L. citreum NRRLB-742.

FIG. 11 shows HPAEC-PAD chromatograms of (1) a mixture of standards; and(2) the New Classic (NC) composite product described in Example 1,wherein the components are A: D-mannitol; B: L-arabinose (internalstandard); C: D-glucose; D: D-fructose; E: sucrose; F: maltose; G-Mrefer to MIMO DP 3-9, respectively; and N-Q refer tomaltooligosaccharides [α-(1,4)] DP 3-6, respectively.

FIG. 12 shows HPAEC-PAD chromatograms of the (1) A2, and, (2) NCcomposite products wherein the components are A: D-mannitol; B:L-arabinose (internal standard); C: D-glucose; D: D-fructose; E:sucrose; F: maltose, G-M refer to MIMO DP 3-9, respectively. Note largerrelative peak areas for DP>4 in the NC composite product.

FIG. 13 shows the carbohydrate components, via high pressure liquidchromatography-refractive index detection (HPLC-RID) of (1) Standardmixture; (2) A2; and (3) NC composite products wherein the componentsare A: MIMO DP>3; B: MIMO DP 3; C: maltose; D: D-glucose; E: D-mannitol;F: maltotriose; and, G: D-fructose.

FIG. 14 shows the second half of the HPLC-RID overlay shown in FIG. 13magnified by a factor of about 10, of (1) Standard mixture; (2) A2; and(3) NC composite products wherein the components are H: lactic acid; I:glycerol; J: acetic acid; K: formic acid; L: propionic acid; and M:isobutyric acid.

FIG. 15 shows an overlay of 1D 300 MHz ¹H NMR (water suppressed in D₂O)spectra of chemical shift reference materials including (1) D-panose(MIMO-DP3); (2) isomaltotriose; (3) maltotriose; and (4) D-glucose,anomerically equilibrated, wherein signal A corresponds to α-(1,4)anomeric protons; B corresponds to α-(1,6) anomeric protons; and C, Dcorrespond to the α and β anomeric protons, respectively, at thereducing end.

FIG. 16 shows an overlay of 1D 300 MHz ¹H NMR (water suppressed in D₂O)spectra of the anomeric regions of 1 and 2, NC and A2 compositeproducts, respectively and, 3, D-panose (δ reference) wherein signal Acorresponds to α-(1,4) anomeric protons; signal B corresponds to α-(1,6)anomeric protons; signals C and D correspond to the α and β anomericprotons, respectively, at the reducing end; and signals E and Fcorrespond to the α-(1,3) and α-(1,2) anomeric protons, respectively.

FIG. 17 shows an overlay of 1D 300 MHz ¹H NMR (water suppressed in D₂O)spectra of the non-anomeric regions of (1) A2 and (2) NC compositeproducts.

FIG. 18 shows an overlay of 1D 300 MHz ¹H NMR (water suppressed in D₂O)spectra of the alkyl regions of (1) NC and (2) A2 composite productswherein signal A is unknown, B corresponds to acetyl protons (thedifference is due to pH, A2=pH 4.81, NC=pH 6.57), C corresponds tolactate C3 methyl protons; and D is unassigned. Note, the amount ofacetate/lactate in 2 (A2) was below the minimum detection limit viaHPLC-RID and barely detectable in the NC composite material.

FIG. 19 graphically illustrates the generation of chemical species (asdetected by HPAEC-PAD and HPLC-RID) throughout the course of a 10 Lfermentation (sucrose:maltose (S/M, w/w) ratio=2.00) with L. citreumNRRL B-742.

FIG. 20 graphically illustrates evolution of the mass average molecularweight distribution (MWD) of MIMOs throughout the course of a 10 Lfermentation (sucrose:maltose (S/M, w/w) ratio=2.00) with L. citreumNRRL B-742. Note that the MWD continues to increase (until about 15hours) after the sucrose is exhausted (at about 10 hours). The rate ofchain growth then takes place at a lower, but constant rate until theend of fermentation (55 hours) when a MWD of 642.5 Da was achieved.

FIG. 21 graphically illustrates the generation of chemical species (asdetected by HPAEC-PAD and HPLC-RID) throughout the course of a 10 Lfermentation (sucrose/maltose=2.75) with L. citreum NRRL B-742.

FIG. 22 graphically illustrates the evolution of the mass averagemolecular weight distribution (MWD) of MIMOs throughout the course of a10 L fermentation (S/M=2.75) with L. citreum NRRL B-742. Note that theMWD continues to increase (until ˜15 hours) after the sucrose isexhausted (˜10 hours). The rate of chain growth then takes place at alower, but constant rate until the end (55 hours) where a MWD of 760.7Da was achieved.

FIG. 23 sucrose:maltose (S/M, w/w) ratio behavior of pH and opticaldensity (OD₆₀₀) through the log-growth phase of a 300 L seedfermentation (sucrose/maltose=2.00, lot #150622) with L. citreum NRRLB-742. Note that pH is not controlled, the culture reachedlate-log/stationary phase, and the final OD was approximately 2.8.

FIG. 24 graphically illustrates the behavior of pH and OD through thelog-growth phase of a 3000 L fermentation (sucrose/maltose=2.75, lot#150622) with L. citreum NRRL B-742. Note that pH was controlled tomaintain 5.50 and that the ultimate OD₆₀₀ was about 10.2.

FIG. 25 graphically illustrates the generation of chemical species (asdetected by HPAEC-PAD and HPLC-RID) throughout the course of a 3000 Lfermentation (sucrose/maltose=2.75, lot #150622) with L. citreum NRRLB-742.

FIG. 26 graphically illustrates the evolution of the mass averagemolecular weight distribution (MWD) of MIMOs throughout the course of a3000 L fermentation (sucrose/maltose=2.75, lot #150622) with L. citreumNRRL B-742. Note that the mass average molecular weight distributioncontinues to increase (until about 15 hours) after the sucrose isexhausted (at about 10 hours). The rate of chain growth then takes placeat a lower, but constant rate until the end (55 hours) where a massaverage molecular weight distribution of 789.5 Da was achieved.

FIG. 27 shows a HPAEC-PAD chromatogram of product lot #150622 whereinthe components are identified as A: D-mannitol; B: L-arabinose (internalstandard); C: D-glucose; D: D-fructose; E: sucrose; F: maltose; andwhere G-M correspond to MIMO DP 3-9.

FIG. 28 shows HPAEC-PAD chromatograms during removal of mannitol frommother liquor 1, and 2, 3 corresponding to compound crystallizationstages, 1 and 2, respectively. The components are identified as A:D-mannitol; B: L-arabinose (internal standard); C: D-glucose; D:D-fructose; E: sucrose; F: maltose; and G-M corresponding to MIMO DP3-9. Note that mannitol is reduced and that the MIMO purity increasedthereby.

FIG. 29 shows an overlay of 1D 300 MHz ¹H NMR (water suppressed in D₂O)spectra of the anomeric regions of 1 and 2, NC and lot #150622 products,respectively and, 3, D-panose (6 reference) wherein signal A correspondto α-(1,4) anomeric protons; signal B corresponds to α-(1,6) anomericprotons; signals C and D correspond to the α and β anomeric protons,respectively, at the reducing end; and signals E and F correspond toregions corresponding to α-(1,3) and α-(1,2) anomeric protons,respectively. Note the minimal or absent second doublet in lot #150622;this is likely due to the acidification step, which avoids possiblealkali catalyzed epimerization of C2.

FIG. 30 shows an overlay of 1D 300 MHz ¹H NMR (water suppressed in D₂O)spectra of the non-anomeric regions of 1) NC and, 2) lot #150622products.

FIG. 31 shows an overlay of 1D 300 MHz ¹H NMR (water suppressed in D₂O)spectra of the alkyl regions of (1) lot #150622; and (2) A2 products.Signal A is an unknown; signal B corresponds to acetyl protons (thedifference is due to pH, A2=4.81, lot #150622=6.08); signal Ccorresponds to lactate C3 methyl protons; and signal D is unassigned.Note, the amount of acetate/lactate in both 1 and 2 (A2) were below theminimum detectable limit (MDL) of HPLC-RID.

FIG. 32 shows the amounts of chemical species as detected by HPAEC-PADand HPLC-RID throughout the time course of a 3000 L fermentation(S/M=2.75, lot #151105) using L. citreum NRRL B-742.

FIG. 33 illustrates the evolution of the mass average molecular weightdistribution of MIMOs throughout the course of a 3000 L fermentation(S/M=2.75, lot #151105) with L. citreum NRRL B-742. Note that the MWDcontinues to increase (until about 15 hours) after the sucrose isexhausted (at about 10 hours). The rate of chain growth then takes placeat a lower, but constant rate until the end of fermentation (55 hours)when the molecular weight of about 776.5 Da was achieved.

FIG. 34 shows HPAEC-PAD chromatograms of (1) product lot #150622, and,(2) Product lot #151105, wherein the components are identified as A:D-mannitol; B: L-arabinose (internal standard); C: D-glucose; D:D-fructose; E: D-leucrose, F: sucrose; G: maltose; and where H—Ncorrespond to MIMO DP 3-9, respectively. For example, note the highproportion of MIMO DP5 (J), DP6 (K), and DP7 (L).

FIG. 35 shows an overlay of HPAEC-PAD chromatograms comparing the (5)ISOThrive product MIMO lot #150622 with commercial MIMO-based products,(1) IMO powder (Bioneutra VitaFiber); (2) IMO powder (BaolingbaoIMO-900); (3) IMO powder (TopHealth AdvantaFiber 90); and (4) IMO powder(Solabia Bioecolians). The components of these compositions wereidentified as A: D-mannitol; B: L-arabinose (internal standard); C:D-glucose; D: D-fructose; E: isomaltose; F: sucrose; G: maltose; and H—Ncorresponding to MIMO DP 3-9, respectively. Note the presence of 30-45%of isomaltose in IMOs 1, 2, and 3. The MWDs were cut off sharply at DP 5for IMO 1. There was an almost undetectable trace of MIMO DP 6 in IMOs 2and 3. Note also, the presence of extensive branching in IMO 4 that isnot observed in the composition generated as described herein.

FIG. 36 is a schematic diagram of a method of manufacturing MIMO.

FIG. 37A-37B graphically illustrate inhibition of colon cancer cellgrowth by media obtained from culture of Lactococcus lactis andLactobacillus gasseri using a CyQUANT NF cell proliferation assay.Dosing was 0 (control), 100, 200, 400, and 800 μg/mL equivalent protein(BCA assay) in both cases. FIG. 37A shows that broth from Lactococcuslactis and Lactobacillus gasseri cultures reduced HCT-15 colon cancercell proliferation in a dose-dependent manner relative to controlculture media. Proliferation of cells was reduced by 60% at the maximumdose tested indicating a dose of appx. 1.3μ g/g equivalent nisin A.Broth produced using L. gasseri demonstrated a clear threshold of effectequivalent to appx. 0.65 μg/g nisin A. FIG. 37B shows that broth fromLactococcus lactis and Lactobacillus gasseri cultures reduced DLD-1colon cancer cell proliferation in a dose-dependent manner relative tocontrol culture media. Antiproliferative activity was similar to thatobserved vs. HCT-15 cells. Again, broth produced with Lactobacillusgasseri demonstrated a dose threshold of approximately half of theequivalent dose of Nisin A. The left-most bar in each cluster showscancer cell growth with no intervention. The second bar (from the left)shows cancer cell growth in the presence of 100 μg/ml of interventionbroth indicated as either from Lactococcus lactis or Lactobacillusgasseri. The third bar (from the left) shows cancer cell growth in thepresence of 200 μg/ml control broth. The fourth bar (from the left)shows cancer cell growth in the presence of 400 μg/ml control broth. Thefifth bar (from the left or the rightmost bar) shows cancer cell growthin the presence of 800 μg/ml control broth.

FIG. 38 graphically illustrates growth of Weissella viridescens in thepresence of known amounts of nisin A (black lines) and nisin Z (graylines). The dashed lines illustrate regression analysis to provide anapproximation of the minimum inhibiting concentrations of nisin A (blackdashed line) and nisin Z (gray dashed line) for inhibiting Weissellaviridescens growth.

FIG. 39 shows an example of a MIMO composition prepared as describedherein (see, e.g., Example 7).

FIG. 40 shows an example of a MIMO composition prepared as described inExample 3.

FIG. 41 shows an example of a MIMO composition prepared as described inExample 4.

FIG. 42 shows an example of a MIMO composition prepared as described inExample 6.

DETAILED DESCRIPTION

Compositions described herein contain maltose-containingoligosaccharides, referred to as maltosyl-isomaltooligosaccharides(MIMOs) that are a preferred energy source of various beneficialintestinal bacteria (e.g., Lactobacillus and/or Bifidobacterium spp.)over other types of oligosaccharide prebiotics. The compositionsdescribed herein can foster the growth and activities of such beneficialintestinal bacteria. Because certain types of beneficial bacteria (e.g.,L. lactis strains) can produce various types of bacteriocins (e.g.,nisins) that can act as anti-cancer agents and/or as anti-microbialagents, the compositions described herein can help to reduce thesymptoms or the incidence of some type of diseases and conditions. Forexample, early studies indicate that the compositions described hereincan reduce or eliminate symptoms in 81% of users who self-identified ashaving acid reflux symptoms once a week or more.

The maltosyl-isomaltooligosaccharides in the compositions describedherein include glucose residues linked mostly by α-(1,6) linkages, andone or two maltose resides (e.g., at the reducing end) that can belinked to a glucose unit by an α-(1,4) linkage. The majority of thelinkages between the glucose units in themaltosyl-isomaltooligosaccharides in the compositions are α-(1,6)linkages, although small numbers of α-(1,2), α-(1,3), and α-(1,4)linkages can be present. The maltosyl-isomaltooligosaccharides aretherefore typically linear oligosaccharides, with few branch points. Themaltosyl-isomaltooligosaccharides terminate in a maltose unit. The massaverage molecular weight distribution (MWD) of the MIMOs in thecompositions described herein can vary depending upon the degree ofpolymerization (DP). For example, the maltosyl-isomaltooligosaccharidescompositions can contain a mass average molecular weight distribution ofabout 520 to 1200 daltons, or of about 640 to 1000 daltons. In somecases, the maltosyl-isomaltooligosaccharides compositions contain a massaverage molecular weight distribution of about 730 to 900 daltons.

An example of an MIMO with a single maltosyl linkage [—O-α-(1,4)-] atthe reducing end, and a DP of 5, can have the following chemicalstructure:

The MIMOs in the compositions described herein can have a number ofglucose units. For example, the MIMOs in the compositions describedherein can have from about 2 to about 18 glucose units, or about 2 toabout 17 glucose units, or about 3 to about 16 glucose units, or about 3to about 15 glucose units, or about 3 to about 14 glucose units, orabout 3 to about 13 glucose units, or about 3 to about 12 glucose units.In general, the maltose-containing oligosaccharides have no more thanabout 17 glucose units, or no more than about 16 glucose units, or nomore than about 15 glucose units, or no more than about 14 glucoseunits, or no more than about 13 glucose units, or no more than about 12glucose units, or no more than about 11 glucose units, or no more thanabout 10 glucose units, for example, as detected by HPAEC-PAD orHPLC-RID.

As indicated, the MIMOs in the compositions described herein can havesmall numbers of α-(1,2), α-(1,3), and α-(1,4) glucosyl linkages. Hence,the MIMOs in the compositions described herein can have small numbers ofbranch points. For example, the MIMOs in the compositions describedherein can have 0-4 branch points, or 0-3 branch points, or 0-2 branchpoints, or 0-1 branch points.

Oligosaccharides and Methods of Making them

Oligosaccharides are an extremely diverse class of molecules, consistingof short chains of monosaccharide molecules composed of less thaneighteen, less than sixteen, less than fourteen, less than twelve, orless than ten monosaccharide units. The monosaccharides observed inprebiotic oligosaccharides are typically galactose(galactooligosaccharides, GOS), glucose (glucooligosaccharides, GlcOS),xylose (xylooligosaccharides, XOS), and fructose(fructooligosaccharides, FOS). GlcOS are a class of carbohydrateoligomers that include isomaltooligosaccharides (IMO). IMOs are glucosylsaccharides with a core structure based on an α-(1→6) linked backbonethat can include α-(1→4), α-(1→3) (nigerooligosaccharides) and\orα-(1→2) (kojioligosaccharides) linked branches (Yun, J. et al.,Biotechnol. Lett., 1994, 16:1145-1150). These glucosidic linkages aretypically found in commercial IMO syrups (Goffin, D. et al., Crit. Rev.Food Sci. Nutr. 51:394-409 (2011)).

Oligosaccharides can be sourced from plants (FOS from agave, yacon;inulin/FOS from chicory), obtained from hydrolyzed starch orexopolysaccharide (e.g. dextran, GlcOS, IMO, MIMO), made viatransglycosylaton via immobilized enzymes, or generated via fermentationproducts conforming to the species-specific exopolysaccharide (EPS) inthe presence of an acceptor molecule (MIMO). Isomaltooligosaccharides,or IMOs, are oligodextrans resulting from the homopolymerization ofglucose, and are thus a distinct sub-class of glucooligosaccharides(GlcOS).

However, the types of MIMOs provided herein are not made by plants.Hence, the MIMOs described herein are not found in foods that aretypically ingested by humans. Instead, the MIMOs described herein aremade under controlled manufacturing conditions by specific strains ofbacteria that express unique types of enzymes needed to provide thestructural attributes of the subject MIMOs.

Dextran can, for example, be made by certain enzymes. For example, mostLeuconostoc species express a glucosyltransferase enzyme (or cohort ofenzyme isoforms) known as dextransucrase. This enzyme, in the presenceof sucrose performs two functions. First, the enzyme cleaves sucrose andexpels fructose. Then, the enzyme cleaves another sucrose molecule andthe glucose unit from such cleavage is glycosidically linked to thefirst glucose. This goes on until very long (>10 kDa up to about 40 MDa)α-(1,6) polyglucan chains, known as “dextran” result.

If dextran is treated with a dextranase enzyme, an α-(1,6) glucosidase,IMO, can result that will have the branched linkages intrinsic to thedextransucrase produced by the parent organism including, for example, adegree of polymerization (DP) or 2-10, and 15% α-(1,3) linkages (whenusing L. mesenteroides NRRL B-1426; Kothari and Goyal, 2015).

In some cases, controlled treatment of dextran from L. mesenteroidesB-512F would yield a distribution of linear (unbranched) α-(1,6)molecules (oligodextrans) and similar treatment by L. citreum B-1299would yield dextran with a distribution of α-(1,2) branched α-(1,6)molecules (branched oligodextrans). It would be possible to mix dextrantypes prior to hydrolysis, as well.

Dextransucrase, however, will, in the presence of sucrose and anacceptor molecule (such as maltose), create MIMOs. The typical route todextran synthesis via dextransucrase is then “interrupted” by theacceptor molecule causing the growth of the chain to terminateprematurely. In effect, the presence of an acceptor such as maltoselimits the molecular weight of the oligosaccharide. When this occurs,specifically with maltose, short glucose chains terminated with maltosewill result. These molecules are referred to as panose-typemaltosyl-isomaltooligosaccharides.

Treatment of starch with enzymes, such as amylase/neopullalanase, eithertogether or sequentially, was noted by Kuriki, et al. (1993) to yieldpanose (MIMO DP 3), isopanose, and maltoooligosaccharides in the rangeof 3 to about 5 (weighted heavily on DP 3), can yield a distribution ofresistant branched molecules (dextrin). Sakano, et al. (1978) noted thattreatment of pullulan with isopullalanase have also been found toproduce panose (MIMO DP 3).

As noted, dextransucrase is a glucosyl transferase that catalyzes thetransfer of a glucose residue from sucrose to a growing polyglucanchain. Many species of bacteria, Leuconostoc spp. in particular, produceone or more of these enzymes, and that the branching pattern unique tothe dextran thus formed is strain-dependent. Indeed, the branchingpatterns of the dextran formed by an organism can be used to speciate atstrain level. Hence, the type of enzyme or microbe used to generate anoligosaccharide can significantly influence the ultimate structure ofthe oligosaccharide mixture produced.

There are two broad varieties of dextransucrases (each having one ormore isoforms). First, there is the soluble fraction, which can beisolated from centrifuged broth (supernatant). Second, there is afraction that contains “insoluble” or “low soluble” dextransucrasebecause it is bound to the cell wall as a dextran complex. Responsiblefor the formation of capsular extracellular polymeric substances (EPS),this insoluble dextransucrase establishes the layer needed forconversion of sucrose to dextran in bulk media, and takes approximatelytwo hours to form after being expressed by the presence of sucrose inthe media (Brooker, 1977). Once formed, dextran forms a thick capsulearound the cell. When this occurs in bulk mixtures, thick andrecalcitrant biofilms are formed. The distinction between soluble andinsoluble dextransucrase is important because the two can producedextrans with different molecular weight distributions and differentlinkage patterns. This is why, besides conformational distortion of theactive site(s), immobilized enzymes behave differently than those in abulk fermentation.

Mechanically, dextransucrase enzymes can be the primary synthetic“reagent” for production of oligosaccharides, including MIMOs. Forexample, Leuconostoc mesenteroides NRRL B-512FMC can produce >95% linearα-(1,6) polyglucan from sucrose.

Metabolically, Leuconostoc spp. typically utilize carbohydrates(sucrose, glucose, and fructose) via heterolactic fermentation, (Dols,et al. 1997), for example, as shown in FIG. 1. In all suchmicrobially-mediated production processes, some of the sucrose istransported into the cell for metabolic purposes, explaining why yieldof dextran and or oligosaccharides produced is limited to approximately50-55%/total sucrose. This also indicates that the competing rates ofmetabolism and dextran production can impact the outcome of afermentation.

The expected fermentation stoichiometry is given below for aerobic andmicroaerobic conditions.D-glucose+O₂→0.92lactic acid+0.95acetic acid+CO₂D-fructose+0.58O₂→0.28mannitol+0.71lactic acid+0.72aceticacid+0.72CO₂  AerobicD-glucose→0.93 lactic acid+0.92 ethanol+CO₂D-fructose→0.53 mannitol+0.56 lactic acid+0.57 acetic acid+0.57CO₂  Microaerobic

Mixtures of isomaltooligosaccharides are generally produced by theaction of immobilized enzymes on mono- or disaccharide feedstocks.Isomaltooligosaccharides can also be produced by transglycosylation ofstarch hydrolysates followed by chromatographic separation. An earlyprocess, Chludzinski et al. produced branched isomaltooligosaccharidesusing dextransucrase (EC 2.4.1.5) expressed from bacterial cultures suchas Leuconostoc spp. and Streptococcus ssp. (Chludzinski, A. M., et al.,J. Bacteriol. B:1-7 (1974)). Roper and Koch later disclosed theproduction of isomaltooligosaccharide mixtures from starch hydrolysates(maltose and maltodextrins) through the action of the α-transglucosidase(EC 2.4.1.24) from Aspergillus spp. (Starch, 1988, 40:453-459).

Further differentiation within the MIMO-class involves the addition ofglucosyl branches. As illustrated herein, such a branch can be just asingle monosaccharide in length. These can occur at the α-(1,2),α-(1,3), and α-(1,4) positions, typically closer to the non-reducing endof the oligosaccharide chain.

The branching patterns are unique to the dextransucrase cohort expressedby a particular bacterial species, and vary tremendously amongstdifferent bacterial strains. For example, while Leuconostocmesenteroides NRRL B-512F creates dextran EPS that is almost completelyunbranched, consisting of >95% α-(1,6) linkages, the dextran produced byL. mesenteroides NRRL B-1299 contains a distribution of three branchedoligomers per DP [roughly 65% α-(1,6), 22% α-(1,2), and 12% α-(1,3)],and L. citreum NRRL B-742 produces a dextran that is somewhere inbetween and can contain 5%-15% α-(1,3). The differences may in the caseof NRRL B-512F be attributed to a single sucrose glucan transferaseenzyme that is responsible for α-(1,6) linkages, and in the case of NRRLB-1299 the differences may be dues to at least three enzyme isoforms(Dols, et al. Appl Environ Microbiol 64(4): 1298-1302 1998). B-1299 isalso capable of expressing glucose glucosyltransferase and fructoseglucosyl transferase when grown on glucose and fructose medium,respectively (id.). These enzymes may create different branchingpatterns, as well. The differences are illustrated in FIG. 2.

The inventors have also observed that the same enzyme cohort (producedby Leuconostoc mesenteroides subsp. mesenteroides (Tsenkovskii) vanTieghem ATCC® 11449™ NRRL B-1299) produces a different branching patternwhen immobilized than when it is native in free solution; the resultswere clear for DP 5 oligosaccharides.

Both branching pattern and molecular weight can have a profound effecton the selectivity of a prebiotic composition. For example, Hu et al.(2013) noted that while Lactobacillus reuteri consumed shorter-chainMIMO, the longer chains (higher molecular weight) were preferred byBifidobacteria. Further, which bacteria will eat a given prebioticoligosaccharide depends on the glycolytic enzyme cohort expressed bythat bacterial type. For example, the MIMO contained in the compositiondescribed herein [>80% linear α-(1,6); also called ISOThrive™], can befermented by Lactobacillus casei NRRL B-1922. Evidence of this isprovided in FIG. 3 where ISOThrive™ composite A2 (see Example #1)material is depolymerized to D-glucose and D-maltose. The compositionsdescribed herein (that include MIMOs such as those in ISOThrive™) canthus be characterized as prebiotics for Lactobacillus casei NRRL B-1922,which is representative of a probiotic bacterial class that is bothlarge and popular.

By means of comparison with a composition containing a highlybranched-type MIMO (produced via NRRL B-1299 dextransucrase), it can bedemonstrated that α-(1,6) linkages are preferred by this organism, andthat α-(1,2), and α-(1,3) linkages are not. This comparison is given inFIG. 4.

It was further noted by Moller, et al (2012) that another popularprobiotic strain, Lactobacillus acidophilus NCFM, preferred MIMO-DP 3(panose) over linear α-(1,6) IMO molecules (DP 3-5). Kothori and Goyal(2012) noted that IMOs produced from NRRL B-1426 dextransucrase (DP2-10, ˜15% α-(1,3) branch linkages) were resistant to the action ofdextranase, α-glucosidase, and α-amylase. Because the α-(1,6)glucosidase is needed for efficient utilization of IMOs by manybacterial species (e.g. L. casei and acidophilus), resistance todextranase tends to rule out the prebiotic potential of this sort of IMOfor most prevalent probiotic lactobacilli strains.

MIMOs conforming to the panose-type structure of oligosaccharides canexist both naturally, or they can be induced by man, in a variety offermented foods (valued as a sweetener rather than a prebiotic)including kimchi with Leuconostoc starter culture fortified with sucroseand maltose (Cho, et al. 2014). In fact, many bacterial species known tosynthesize exopolysaccharides (EPS), e.g. dextran or levan, fromsucrose, have been isolated from sourdough starter cultures. Examplesinclude the following (see Tieking, et al. 2003):

Isolated species, TMW #: EPS type: Monomer: L. sanfranciscensis spp.Levan Fructose L. frumenti spp. Levan Fructose L. pontis spp. LevanFructose L. panis 1.649 Levan Fructose L. reuteri 1.106 Dextran GlucoseL. reuteri 1.109 Reuteran Glucose W. confusa 1.617 Dextran Glucose W.confusa 1.934 Levan Fructose

Other facultative anaerobic heterofermentative organisms have also beennoted including (Corsetti and Settanni, 2007) Leuconostoc, Weissella,Pediococcus, Lactococcus, Enterococcus, and Streptococcus spp. have beenisolated from sourdough.

As shown by the inventors, all but Enterococcus spp. (which produceheteropolymeric EPS) produce either dextran or levan (see also, Mozzi,et al. 2006). The inventors have shown that some MIMO is present in aspontaneous sourdough fermentation of red wheat flour, where it wasnoted to arise from sucrose in the flour and maltose from the starch.See, e.g., FIG. 5. Subsequent analysis revealed that the starter culturewas Pediococcus pentosaceus.

The bacterial strains present in the kimchi (beginning and end offermentation) were determined via high-throughput PCR and sequencing ofthe 16S rRNA; the results are given here:

Sample ID Species Kimchi Juice NR 102781 Leuconostoc carnosum JB16 6.8%NR 074997 Leuconostoc gasicomitatum LMG 42.1%  NR 102984 Leuconostocgelidum JB7 12.2%  NR 025204 Leuconostoc inhae strain IH003 2.6%

Recently, use of the enzyme glucosyltransferase isolated fromLeuconostoc mesenteroides has been reported by Remaud et al., whodisclosed production of linear and short-branched oligosaccharides withα-(1→6) linkages and a maltose at the reducing end (Remaud, M., et al.,J. Carbohydrate Chem., 1992, 11(3):359-378). Remaud et al. used afeedstock starting ratio of 7:1 sucrose:maltose in a reaction catalyzedby extracellular Leuconostoc mesenteroides ATCC 13146glucosyltransferase that produced branched dextrans with α-(1-3)linkages (id.). The same group has reported the production of branchedα-(1,2) isomaltooligosaccharide mixtures from sucrose with an acceptorreaction catalyzed by dextransucrase (Remaud-Simeon, M. et al., Appl.Biochem. Biotechnol. 1994, 44:101-17).

Paul et al. disclosed the synthesis and purification of branchedisomaltooligosaccharide mixtures containing an α-(1,2) bond by theaction of soluble and insoluble glucosyltransferase isolated fromLeuconostoc mesenteroides B-1299 on sucrose and a glucosyl acceptor suchas maltose (or a material rich in maltose, such as a starch hydrolysisproduct), isomaltose, methyl α-glucoside, isomaltotriose or glucose (ora material rich in glucose, such as a starch hydrolysis product) (Paul,F. et al., U.S. Pat. No. 5,141,858).

D-fructose, the byproduct of transglucosylation via sucrose, is adifficult compound to separate from the isomaltooligosaccharide product,and can be a detriment to use of this product in human nutrition, giventhe current information about the negative effects of high fructosesyrups. (See: Ouyang, X., et al., J. Hepatol., 2008, 48(6):993-9. doi:10.1016/j.jhep.2008.02.011. Epub 2008 Mar. 10; Dhingra, R. et al.,Circulation, 2007, 116:480-488; Swanson, J. E., et al., Am. J. Clin.Nutr., 1992, 55(4):851-856; and Vartanian, L. R., et al., Am. J. PublicHealth, 2007, 97(4):667-75. Epub 2007 Feb. 28.)

Preferred Methods of Producing MIMOs Compositions

On a commercial scale, the composition of the present invention can beproduced as described in U.S. patent application Ser. No. 14/833,094,filed Aug. 22, 2015, and entitled “Process for the Production ofIsomaltooligosaccharides,” which is herein incorporated by reference inits entirety.

A process flow diagram of the manufacturing process is provided in FIG.36. In general, the process consists of two steps including, first, theenzymatic synthesis of MIMO via extracellular dextransucrase expressedby a suitable organism during the fermentation, in rich mediumcontaining sucrose, and second, downstream processing to remove biomass,residual salts/minerals, and bacterial metabolites.

Certain metabolites, particularly D-mannitol, can remain in the MIMOproduct composition and positively affect its organoleptic character.

The compositions described herein can have a pH that is sufficiently low(1.5 to 2.8 pH, 1.5 to 4.2 pH, or 2.0 to 4.2 pH) to preventmicrobiological spoilage and or pathogens while improving theorganoleptic character of the composition. For example, the product pHcan in some cases be pH 1-7, or pH 1-6, or pH 1-4, or pH 1-3, or pH1.5-3.0, where pH 1.5-2.8 is preferred. A variety of acidulants may beadded including, but not limited to, organic acids including citric,malic, lactic, tartaric, fumaric, succinic, ascorbic, benzoic, adipic,caprylic, propionic, acetic acids, and salts (mono, di- and tri-Na, Mg,Ca, etc.), stereoisomers (L/D, R/S, meso), rotamers, esters, andmixtures thereof, and mineral acids including phosphoric, hydrochloric,sulfuric, and salts, esters, and mixtures thereof, and amino acids,including lysine, cysteine, methionine, glutamic, and aspartic acids,and salts, esters, and mixtures thereof.

In addition, the composition can in some cases be concentrated to asufficiently high water activity (aW) to protect the shelf life.

Hence, in some cases, the low amounts of water and the low pH canprotect the composition from spoilage or microbial growth.

The mass average molecular weight distribution (MWD) of the MIMO tendsto increase with sucrose:maltose (S/M) ratio present at the time ofinoculation (see FIG. 6). The relationship between sucrose:maltose ratioand the MWD of the product can be approximated (±6%, R²=0.9715, N=29)as: MWD, Da=151.56*S/M+358.58. Note that although a previous U.S. patentapplication Ser. No. 14/833,094, filed Aug. 22, 2015, mentions that asthe S/M increases so does the molecular weight distribution, thatprevious application did not provide a mathematical relationship betweensucrose:maltose ratio and the MWD of the product. Further studies havebeen performed, and the relationship between sucrose:maltose ratio andthe MWD of the product has been defined more precisely herein.

The mass average molecular weight distribution is also dependent on thepH optimum of the dextransucrase(s) expressed by the fermentingorganism. For dextransucrases from Leuconostoc spp. (NRRL B-512F, B-742,and B-1299), this optimum is 5.5±0.3 (Miller et al. 1986, Dols-Lafargueet al. 2001). This behavior with respect to pH appears to be conservedwithin enzymes produced by a variety of organisms. For example, the pHoptimum for dextransucrase isolated from Pediococcus pentosaceous is 5.4(Patel, et al. 2011) and that from Streptococcus mutans was optimum at5.5 (Chludzinski et al. 1974). The pH optimum is rather broad, and canbe affected by substrate concentration (Sarwat, et al. 2008), ionicstrength, and the presence of certain cations such as magnesium andcalcium (Patel, et al. 2011). Furthermore, gene deletions modifying thecarboxy-terminus of the active site in dextransucrase from NRRL B-512Faffected neither km nor the pH optimum (Monchois, et al. 1998). Atypical pH optimum curve for the B-512F enzyme is given in FIG. 7, andthe dextransucrase involved with the synthesis of the compositiondescribed herein is typical.

The mass average molecular weight distribution (MWD) is approximated bya function dictated by pH optimum, but is, in the embodied process,attenuated, and, at a sucrose:maltose ratio S/M of 2.00 (approximately2.73 at time of inoculation) appears to have a quadratic maximum betweenpH 5.5 and 6.0 that can be approximated by the following:MWD,Da=−159.1*pH²+1866.1*pH−4688.

Note that although a previous U.S. patent application Ser. No.14/833,094, filed Aug. 22, 2015, mentions that, as pH either increasesabove or decreases below the optimum range for the enzyme, both themolecular weight distribution and MIMO yield decrease, that previousapplication did not provide a mathematical relationship between pH setduring fermentation and the MWD of the product. Further studies havebeen performed, and the relationship between pH and the MWD of theproduct has been defined more precisely herein.

The organisms used herein, including Leuconostoc mesenteroides, arecapable of producing dextransucrase, and can be utilized to produce thecomposition containing maltosyl-isomaltooligosaccharides pursuant to themethods described herein. In another example, L. citreum ATCC 13146(NRRL B-742) may be used. This bacterium is known by other designationsby those skilled in the art, including the designation Leuconostoccitreum ATCC 13146, the designation NRRL B-742, the designationLeuconostoc citreum Farrow, and the designation L. amelibiosum. Thebacterium Leuconostoc mesenteroides subsp. mesenteroides (Tsenkovskii)van Tieghem ATCC® 11449™ (NRRL B-1299) can also be employed. Inaddition, the inventors have also tested L. citreum B-1355, Weissellaconfusa B-1064, and Lactobacillus sanfransiscensis ATCC 27651. TheB-1355 L. citreum B-1355 and Weissella makes MIMO, and can make largerMIMO molecules.

Other useful dextransucrase/alternansucrase-producing microorganismsinclude, but not limited to, Leuconostoc spp (specificallymesenteroides, citreum, gasicomitatum, carnosum, gelidum, inhae, andkimchi), Weissella spp (specifically confusa, kimchi), Lactococcus spp.,Streptococcus spp. (specifically mutans), Lactobacillus spp. (e.g.reuteri), Pediococcus spp. (specifically pentosaceus), and certainmutant E. coli. Useful microorganisms, or mixed cultures thereof, mayalso be isolated from natural sources including, but not limited to,spontaneous (wild) sourdough starter cultures (the bioorganism mixtureused in the production of sourdough bread) and kimchi.

The organism used in the Examples described herein is Leuconostoccitreum Farrow et al. ATCC 13146 (NRRL B-742).

Synthesis

On a commercial scale, the composition of the present invention may beproduced as described in U.S. patent application Ser. No. 14/833,094,filed Aug. 22, 2015, entitled “Process for the Production ofIsomaltooligosaccharides”, which is herein incorporated by reference inits entirety.

In general, upon start-up of the fermentation process, the entireequipment system is flushed, cleaned and sterilized. A fermentation tankis charged with the requisite media components (typical vitamins,sulfates, phosphates, salts and other materials used for bacterialculture, sucrose, and maltose in a defined sucrose to maltose ratio. Allingredients are non-GMO and certified Kosher/Pareve, including thebacterial vial stock. Separately, the inoculum (in the preferredapproach, ATCC 13146) is grown until achieving an optical density of atleast 1 (OD, or absorbance at 660 nm via UV-VIS spectrophotometer), andadded to the fermentation in a volume in the range of about 1% to about10% (1% in the preferred approach) of the amount contained in the bulkmedia. The fermentation takes place at a temperature of about 27° C. andis maintained at pH 4.0-6.0 (5.5 in the preferred approach) via additionof 50% w/w sodium hydroxide. The fermentation is not aerated, but theheadspace is pressurized with air as needed to maintain positivepressure. The fermentation is continued until no fructose is present,for a period of approximately 25 to 60 hours. The characteristics andbehavior of the feedstock, products, and metabolites throughout thecourse of fermentation are given in Examples 3, 4, 6, and 7. Most, ifnot all, of the sucrose and maltose are either consumed or converted toMIMO within a few hours, usually within about 10 hours.

The fermentation is continued for a certain amount of time thereafter toallow the MIMOs produced to be enzymatically rearranged to yield longerchains (higher MWD), if desired. Typically, the time required to createa product, for example, of target MWD of 760-800 Da is about 55 Hr.Ideally, the batch will be run, with periodic sampling and analysis byHPAEC-PAD and/or NMR until the target MWD is reached and the broth isessentially free of fructose. The fermentation would then be terminatedand moved into downstream processing. The behavior of the MWD of MIMOthroughout the course of a 3000 L commercial fermentation is given inExamples 6 and 7.

Downstream Processing

The composition of the present invention may be purified as described inU.S. patent application Ser. No. 14/833,094, filed Aug. 22, 2015,entitled “Process for the Production of Isomaltooligosaccharides,” whichis incorporated herein by reference in its entirety.

During the production of the maltosyl-isomaltooligosaccharidescomposition, the MIMO fermentation can be determined to be complete asdetermined by the conversion of the fructose to mannitol and byachievement of a target molecular weight distribution. In some cases,the MIMO fermentation is determined to be complete when the fructose isconsumed, by conversion of fructose to mannitol, and/or by cessation ofthe take up of alkali. Hence, the amount of fructose in the finalfermentation fluid can be less than less than 4%/brix fructose, or lessthan 3%/brix fructose, or less than 2%/brix fructose, or less than1%/brix fructose, or less than 0.5%/brix fructose, or less than0.25%/brix fructose as detected by HPAEC-PAD or HPLC-RID.

The cells can be separated from the broth by microfiltration,centrifugation, or by other broth clarification processes. The cells arethen discarded.

The broth can be concentrated by evaporation to 35-45 brix anddecolorized with powdered activated carbon (PAC, 0.4-2.0%/broth mass) atabout 60-80° C. Alternatively, either granulated activated carbon (GAC)or powdered activated carbon (PAC) may be used, which is later removedby filtration. In another example, granulated activated carbon (GAC) orpowdered activated carbon (PAC) can be packed into a jacketed column andused for decolorization in a single-pass or multiple-pass operation.

The decolorized liquor is then demineralized and the organic acidmetabolites removed by a two-stage ion exchange process (IEX), wherebythe liquor is contacted first with a strong acid cation (SAC) ionexchange resin, and second, the liquor is contacted with a weak baseanion (WBA) IEX resin.

The resulting liquor (2-10 brix) is adjusted to pH<4.2 (typically2.0-4.0) with phosphoric acid (H₃PO₄, 85%, 0.25-2.00 kg) and isconcentrated by evaporation to 45-60 brix (56 is the preferredapproach). The resulting concentrate is slowly cooled, with gentleagitation, to room temperature and allowed to crystallize.

The resulting liquor is separated from the crystals (D-mannitol) viafiltration (Nutsch) or by basket centrifuge. The crystal cake is washedwith water (100-200% cake mass at 77% to 95% solids, 125-150% ispreferred). The washings are either retained for future batch runs orcombined with the liquor and concentrated by evaporation to 60-70 brix(67-69 brix is preferred).

The resulting concentrate is slowly cooled, with gentle agitation, toroom temperature (19-25° C.) and crystals allowed to seed. Once theinitial target temperature has been reached, the whole is then cooledslowly to 2-10° C. (5° C. is preferred) until crystallization iscomplete.

The resulting liquor is separated from the crystals (D-mannitol) viafiltration (Nutsch) or by basket centrifuge. The crystal cake is washedwith cold water (100-200% cake mass with 77% to 95% solids, 125-150%cake mass at 95% solids is preferred).

The product liquor is pasteurized (70° C. for 30 min.) prior to packout. The washings are retained and frozen for recycle into the nextbatch run as appropriate.

Where the concentration of the components in the composition so preparedwill fall into the ranges established below. The composition may beliquid at 59-68%/brix or a spray dried/lyophilized powder. The MWD canbe inside the range of 730 and 900 Da (mass average).

Minimum Maximum Component: %/brix: %/brix: MIMO DP3-9: 69.00 100.00D-Mannitol: 0.00 14.00 D-Glucose 0.00 2.50 D-Fructose 0.00 0.50 Sucrose0.00 6.00 D-Maltose 0.50 7.00 Lactate 0.00 6.50 Glycerol 0.00 2.50Formate 0.00 1.00 Acetate 0.00 10.00 Purity, %: 69.00 100.00The following composition is prepared on a commercial scale and ispreferred. Syrups are typically between 62 and 65 brix.

Minimum Maximum Component: %/brix: %/brix: MIMO DP3-9 84.77 95.51D-Mannitol 6.23 8.35 D-Glucose 0.46 0.85 D-Fructose 0.05 0.16 Sucrose1.75 1.86 D-Maltose 4.29 4.39 Glycerol 0.41 0.51 Total 98.58 111.03The MIMOs are distributed over a range of DP values between two and ninewith a mass-average molecular weight distribution in the range of730-900 Da:

Minimum Maximum Component: %/brix: %/brix: maltose 0.93 6.83 MIMO-DP34.17 14.89 MIMO-DP4 10.80 31.62 MIMO-DP5 13.04 29.89 MIMO-DP6 8.03 21.03MIMO-DP7 2.90 13.79 MIMO-DP8 1.24 8.25 MIMO-DP9 0.00 3.69Wherein a MWD of 780 Da±1.65% is commercially prepared, and thuspreferred:

Minimum Maximum Component: %/brix: %/brix: maltose 4.29 4.39 MIMO-DP311.89 14.89 MIMO-DP4 22.19 28.06 MIMO-DP5 23.66 28.67 MIMO-DP6 15.2316.77 MIMO-DP7 5.75 6.58 MIMO-DP8 2.48 2.80 MIMO-DP9 0.58 0.73Wherein the composition differs from all other commercial IMO productsby virtue of overall composition (MIMO purity), MIMO MWD, and/orproportion of glycosidic linkage types.

It is understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present application will be limitedonly by the appended claims and their equivalents.

Treatment

The compositions described can be administered in a regimen that fostersthe growth and/or activity probiotic microorganisms that can be presentin the digestive or gastrointestinal system of an animal. For example,the compositions described herein can be administered to animals,including humans, domesticated animals, zoo animals, and wild animals.The compositions can be used routinely or intermittently to foster thegrowth and/or activity of probiotic microorganisms that can be presentin the digestive or gastrointestinal system of an animal.

The compositions contain an amount of MIMOs that can be effective forfostering the growth and/or activity of probiotic microorganisms. Aneffective amount can, for example, be an amount sufficient to fosterprobiotic microorganism growth by at least about 5%, 10%, 20%, 25%, 30%,40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, or 99.9%. In some cases, the population of probioticmicroorganisms can be increased in a gastrointestinal system of ananimal who has received the compositions described herein. For example,the population of probiotic microorganisms can increase by abouttwo-fold, or about three-fold, or about four-fold, or about five-fold,or about six-fold, or about seven-fold, or about ten-fold.

The compositions can be administered or ingested once a day, or twice aday, or three times a day. In some cases, the compositions can beadministered or ingested every day for one week, or for one month, orfor two months, or for three months, or for six months, or for one year,or for two years, or for three years. In many cases the compositions canbe administered or ingested every day indefinitely. The compositions maybe administered in single or divided dosages.

The compositions described herein can include a mixture of MIMOs asdescribed herein. Inactive ingredients can be present in thecompositions such a mannitol and some of the other components describedherein. For example, the MIMOs can be present as about 50%, or about60%, or about 65%, or about 70%, or about 75%, or about 80%, or about85%, or about 90% or about 95%, or about 96%, or about 97%, or about98%, or about 99% of the composition.

The compositions can be administered or ingested in amounts of at leastabout 0.01 mg/kg to about 100 mg/kg, of at least about 0.01 mg/kg toabout 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight,although other dosages may provide beneficial results. For example, thecompositions can be administered or ingested in amounts of at leastabout 0.1 g, or at least about 0.25 g, or at least about 0.5 g, or atleast about 0.7 g, or at least about 0.8 g, or at least about 0.9 g, orat least about 1.0 g, or at least about 1.1 g, or at least about 1.2 g.The unit dosage can vary from about 0.01 g to about 50 g, from about0.01 g to about 35 g, from about 0.1 g to about 25 g, from about 0.5 gto about 12 g, from about 0.5 g to about 8 g, from about 0.5 g to about4 g, or from about 0.5 g to about 2 g.

The amount administered will vary depending on various factorsincluding, but not limited to, what types of compound(s), and/or othertherapeutic agents are administered, the route of administration, theprogression or lack of progression of the disease, the weight, thephysical condition, the health, the age of the patient, whetherprevention or treatment is to be achieved, and if the antigen or ligandis chemically modified. Such factors can be readily determined by theclinician employing animal models or other test systems that areavailable in the art.

Compounds and compositions thereof may be administered in a single dose,in multiple doses, in a continuous or intermittent manner, depending,for example, upon the recipient's physiological condition, whether thepurpose of the administration is therapeutic or prophylactic, and otherfactors known to skilled practitioners. The administration of thecompositions may be essentially continuous over a pre-selected period oftime or may be in a series of spaced doses. Both local and systemicadministration is contemplated.

In some cases, the compositions are formulated as liquid formulations.Alternatively, the MIMO compounds and other ingredients may be in powderform, obtained by aseptic isolation of sterile solid or bylyophilization from solution, for constitution with a suitable vehicle,e.g., sterile, pyrogen-free water, before use.

When the MIMOs are prepared for oral administration, they can becombined with a carrier. Such a carrier can be a pharmaceuticallyacceptable carrier, diluent or excipient. The compositions can beprovided in the form of a unit dosage form. For oral administration, theMIMOs may be present as a powder, a granular formulation, a solution, asuspension, an emulsion or in a natural or synthetic polymer or resinfor ingestion of the active ingredients from a chewing gum. Thetherapeutic agents may also be presented as a bolus, electuary or paste.

In some case, the compositions can be prepared for, and administered as,oral compositions. For example, tablets or caplets containing thecompounds, and optionally a carrier can include buffering agents such ascalcium carbonate, magnesium oxide and magnesium carbonate. Caplets andtablets can also include inactive ingredients such as cellulose,pre-gelatinized starch, silicon dioxide, hydroxy propyl methylcellulose, magnesium stearate, microcrystalline cellulose, starch, talc,titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil,polypropylene glycol, sodium phosphate, zinc stearate, and the like.Hard or soft gelatin capsules containing at least one therapeutic agentof the invention can contain inactive ingredients such as gelatin,microcrystalline cellulose, sodium lauryl sulfate, starch, talc, andtitanium dioxide, and the like, as well as liquid vehicles such aspolyethylene glycols (PEGs) and vegetable oil. Moreover, enteric-coatedcaplets or tablets containing one or more of the compounds of theinvention are designed to resist disintegration in the stomach anddissolve in the more neutral to alkaline environment of the duodenum.

The compositions can also include antioxidants, surfactants,preservatives, film-forming, keratolytic or comedolytic agents,perfumes, flavorings and colorings. Antioxidants such ast-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytolueneand α-tocopherol and its derivatives can be added.

The subjects to whom the compositions can be subjects that may have oneor more diseases or conditions. Examples of diseases or conditions caninclude cancer, pre-cancerous condition(s) or cancerous propensities,diabetes (e.g., type 2 diabetes, or type 1 diabetes), autoimmunedisease(s), acid reflux, vitamin deficiencies, mood disorder(s),degraded mucosal lining(s), ulcerative colitis, digestive irregularities(e.g., Irritable Bowel Syndrome, acid reflux, constipation, or acombination thereof), inflammatory bowel disease, ulcerative colitis,Crohn's disease, gastroesophageal reflux disease (GERD), infectiousenteritis, antibiotic-associated diarrhea, diarrhea, colitis, colonpolyps, familial polyposis syndrome, Gardner's Syndrome, Helicobacterpylori infection, irritable bowel syndrome, and intestinal cancers. Thecompositions can foster the growth and activity of certain types ofbacteria (e.g., L. lactis strains) leads to the production of varioustypes of bacteriocins (e.g., nisins) that can act as anti-cancer agentsand/or as anti-microbial agents. For example, early studies indicatethat the compositions described herein can reduce or eliminate symptomsin 81% of users who self-identified as having acid reflux symptoms oncea week or more.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present application, the preferredmethods and materials are now described.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

“Brix”, also known as degrees Brix (symbol ° Bx), refers to the sugarcontent of an aqueous solution. One degree Brix is 1 gram of sucrose in100 grams of solution and represents the strength of the solution aspercentage by weight (% w/w). Brix also accounts for dissolved salts,organic acids, and other solutes that increase the refractive index ofthe solution. As such, it is less useful as a quantitative measure ofsaccharide content in complex broth (fermentation mixtures), but isquite accurate with respect to the refined product. Thus, 1 degreebrix=1 g refractive dry solids per 100 g of material. If the solutioncontains dissolved solids other than pure sucrose, then the ° Bx onlyapproximates the dissolved solid content. However, when the constituentcomponents of the compositions to be compared are similar and/or withinsimilar ranges, Brix values are reproducible and provide anapproximation which, in this case, is an accurate (relative to true drysolids via evaporation) measurement of relative dry solids per eachcomposition.

“Optical density” or “OD” refers to an estimation of cellular density ina fermentation. Typically used to determine the progress of afermentation, it is determined via absorbance of light at 600 nm and maybe referenced to dry cell mass.

“HPAEC-PAD” refers to a hyphenated instrumental analytical techniqueknown as High Pressure Anion Exchange (HPAEC) liquid chromatography(ThermoDionex ICS-5000+) with a Pulsed Amperometric Detector (PAD).Under the scope of this work, this instrument is used solely for thehigh-resolution separation (ThermoDionex Carbopac PA-100, pH>12.5,acetate gradient elution) of sugar alcohols, mono and disaccharides, andoligosaccharides. Quantification is done via internal standard usingL-arabinose and response factors relative to either the pure compound orto a purified maltodextrin of equivalent molecular weight.

“HPLC-RID” refers to a hyphenated instrumental analytical techniqueknown as High Pressure Liquid Chromatography (HPLC, Agilent 1100) with aRefractive Index Detector (RID). Under the scope of this work, thisinstrument is used to separate (BioRad Aminex HPX-87H, 0.008N H₂SO₄isocratic) and quantify organic (carboxylic) acids that result frombacterial fermentation. This instrument is also used to confirm DP 3,maltose, and mannitol. Quantification is done via external standardmethod vs. a mixed standard made from target compounds of known purity.

“SIP” refers to sterilized in place.

“SBF” refers to sterilization by filtration through a 0.2 μm membrane.

“CIP” refers to cleaned in place.

“DSP” refers to downstream processing.

“PRMXE” refers to filter cartridge series PRMXE or “Pur-Maxx E”dual-layer pleated polyethersulfone membrane sterilizing grade (SG)cartridges; 0.20 μm.

“Degree of polymerization”, or “DP”, refers to the number ofmonosaccharide sugar units in a given oligosaccharide.

“Oligosaccharides” refers to glycans of all kinds with DP>=3 and <=10.

“Molecular weight distribution,” or “MWD” refers to the mass-averagemolecular weight of a distribution of oligosaccharides.

“Oligosaccharides” refers to glycans of all kinds, generally with adegree of polymerization (DP) greater than or equal to 3 and less thanor equal to 18.

“Glucooligosaccharide”, or “GlcOS”, refers to homopolymeroligosaccharides (comprised of glucose in any structural arrangement).GlcOS include the maltooligosaccharide series, typically derived fromplant starch. One example, is [—O-α-(1,4)-], maltopentaose, which hasthe following chemical structure:

“Isomaltooligosaccharide”, or “IMO”, refers to glucosyl saccharides witha core structure based on an α-(1→6) linked backbone that may includeα-(1→4), α-(1→33) (nigerooligosaccharides or kojioligosaccharides),and/or α-(1→4) (maltooligosaccharide)-linked branches. IMO is a GlcOSassembled with a core of [—O-α-(1,6)-] linkages, accordant with thedextran core structure. One example is isomaltopentaose, which has thefollowing chemical structure:

“Maltosyl-isomaltooligosaccharides,” or MIMOs, refers tooligosaccharide, specifically IMO, typically of less than 10-18 degreesof polymerization comprised of α-(1→6) linkages and terminated by anα-(1→4) glucosyl linkage. The α-(1→4) terminal group originates frommaltose. Therefore, maltosyl-isomaltooligosaccharide or MIMO is producedby an acceptor reaction by maltose or other maltooligosaccharide. Anexample of an MIMO with a single maltosyl linkage [—O-α-(1,4)-] at thereducing end, and accordant with the panose core structure, ismaltosyl-isomaltotriose, which has the following chemical structure:

“Branched MIMO” refers to an oligosaccharide, specifically MIMO, of lessthan or equal to 10 degrees of polymerization comprised of α-(1→6)linkages terminated by an α-(1,4) glucosyl linkage and α-(1,2), α-(1,3)and/or α-(1,4) branches. Examples of a branched MIMO with glucosebranching linkages at positions 1,2 and 1,3 and/or 1,4 have thefollowing structures:

“Dietary supplement” refers to a food, food ingredient, or food additivethat produces a health benefit. Carbohydrates such as oligosaccharidescan be dietary supplements.

“SAC” means a Strong Acid Cation exchange resin, typically one withsulfonic acid groups, i.e., a sulfuric acid equivalent. In someinstances, SAC is referring to Purolite C-150S, which has a H⁺ capacityof 1.8 mol/L.

“WBA” means a Weak Base Anion exchange resin, typically one withtertiary amine groups, which are not stronger than the correspondingfree base (pKa˜9.8). In this case, WBA is referring to Purolite A-133which has a free-base capacity of 1.8 mol/L.

The Examples illustrate some of the experimental work performed in thedevelopment of the invention.

Example 1

Two variations of product were used in clinical trials. They varied onlyin molecular weight distribution, and this was controlled by varying thesucrose to maltose ratio (S/M). “A2”-type products have a molecularweight of about 750 Da and “Classic” or “NC”-type products have amolecular weight of about 800 Da. Each of the two types were a compositeof six 16 kg fermentations each of A2 and NC types (12 totalfermentations). The clinical trials were both double-blind placebocontrolled studies, and the parameters for each are given here.

Clinical Trial Protocol

Purpose:

To compare the effects of daily intake of two different formulations ofthe ISOThrive supplement vs. a placebo on the primary outcome measure ofbody weight and secondary outcome measures (inflammatory and satietyserum markers, hunger/satiety, health-related measures and self-reportedquality of life) in a group of overweight but otherwise healthy adults.

Study Design:

A randomized, placebo controlled, double-blind parallel design controltrial to compare the effects of daily intake for a 3-month period of theISOThrive supplement vs. a placebo.

The study design included 3 treatment arms:

-   -   (1) ISOThrive supplement (Type 1, “A2”)    -   (2) ISOThrive supplement (Type 2, “NC”)    -   (3) Placebo supplement (high-maltose syrup at 64 brix)        The two ISOThrive supplements (Type I and 2) have the same        active ingredients, and a dosage of 1000 mg of MIMO. The two        types of the supplement differ in terms of purity (MIMO/total)        and the mass-average molecular weight distribution of the MIMO,        which is the principal ingredient.        Study participants included 105 overweight men and women in the        age range of 18 to 75 years, who are nonsmokers with a body mass        index (BMI) > or =25 and a maximum body weight of 350 pounds        Another Trial Protocol        Purpose:

First, to evaluate, via 16S rRNA sequencing of fecal swabs, the effectof a nutritional supplement (specific soluble fiber known asisomalto-oligosaccharides) on the abundance, diversity, and predictedmicrobiome gene function, of gut bacteria. Second, to evaluate theoverall subject condition in terms of body weight, and self-reported guthealth data. The test groups were compared across-supplement and withthe placebo group.

Study Design:

a randomized, placebo-controlled trial, with a dose escalation at themid-point of the intervention period.

Subjects were between the ages of 18 and 45 years, with a maximum weightof 350 lbs., and a body mass index 25 kg/m² or higher, and have generalself-reported good health.

60 subjects were randomized to three arms (20 each: Supplement type-1,Supplement formula type-2, or placebo) took a daily dose of eitherSupplement A, Supplement B, or placebo for 8 weeks.

Doses included 500 mg of MIMO during the first 4 weeks and then included1000 mg of MIMO for a second 4 weeks.

Composition for Clinical Trials: “A2” and “Classic” Type Products

Six fermentations were carried out to generate each type of productusing a 20 L fermenter (New Brunswick BioFlo 410), that was charged withthe following medium:

“A2” Type “Classic” Type N = 6 Kg: Stdev: RSD, %: N = 6 Kg: Stdev: RSD,%: Water 12.594 0.0144 0.114 Water 12.600 0.0004 0.003 Sucrose 1.8000.0000 0.000 Sucrose 1.910 0.0004 0.021 Maltose-H₂O 0.998 0.0000 0.000Maltose-H₂O 0.864 0.0000 0.000 MnSO₄-H₂O 0.0002 0.0000 1.000 MnSO₄-H₂O0.0002 0.0000 1.610 MgSO₄ 0.0015 0.0000 0.209 MgSO₄ 0.0015 0.0000 0.281FeSO₄-7H₂O 0.0002 0.0000 1.454 FeSO₄-7H₂O 0.0002 0.0000 0.801 KH₂PO₄0.0400 0.0000 0.014 KH₂PO₄ 0.0400 0.0000 0.076 NaCl 0.0002 0.0000 0.824NaCl 0.0002 0.0000 1.058 CaCl₂-2H₂O 0.0008 0.0000 0.390 CaCl₂-2H₂O 0.0010.0000 1.479 Yeast Extract 0.075 0.0004 0.543 Yeast Extract 0.076 0.00081.108 NaOH, 50% 0.016 0.0003 2.056 NaOH, 50% 0.016 0.0007 4.379 Total:15.53 0.040 0.25 Total: 15.51 0.0232 0.15 TS, %; 17.38 0.042 0.24 TS, %;17.33 0.0259 0.15 Brix, %: 18.19 0.031 0.17 Brix, %: 18.15 0.0313 0.17S/M: 2.00 0.000 0.00 S/M: 2.45 0.0005 0.02The pH of the medium was adjusted to 7.00 with NaOH (50%).

200 mL of the medium was transferred to an Erlenmeyer flask, sealed andautoclaved at 121° C. for 15 minutes, cooled, and inoculated with 1 mLvial stock (0.5 mL late-log culture in 20% glycerol, stored at −75° C.).The seed was incubated with swirling at 27° C. and allowed to grow forabout 16 hr. The balance of the medium was transferred to the fermentervia sanitary pump. The fermenter was sealed and sterilized in place(SIP) to 116° C., then rapidly cooled to 30° C. (see temperature curvein FIG. 8). The curve was modeled, and the model was used to determinethe time spent in each microbiologically relevant regime. The resultswere as follows:

T ° C.: Hr: >70 2.43 >80 2.22 >90 1.86 >100 1.16 >110 0.46

These results are relevant because when the full media is mixed andadjusted to pH 7.00 (to avoid inversion of sucrose) significant amountsof maltose (a reducing sugar) are lost via the Maillard reaction. Inaddition to generating a great deal of colored material (>10,000 IU),this increases the S/M of the medium (as detected by HPLC-RID), and hasthe effect of increasing the ultimate molecular weight distribution ofthe product.

The fermenter was inoculated with 150 mL of the flask seed culturecontaining Leuconostoc citreum ATCC 13146 (NRRL B-742) and the pH of themedium was corrected to 6.50 using a solution of 37% HCl. Thefermentation was allowed to proceed for 55 Hr with pH adjustment using asolution of 40% NaOH to maintain the pH at 5.50.

Typically, there is a 4-6 hour induction period, or lag-phase, precedinglog-growth. The log-growth phase is about 10 hours (where most growthoccurs) but it typically takes about 18 hours to reach stationarygrowth. pH 5.5 is typically achieved within about 2 hours of log-phasegrowth, and this pH is maintained thereafter. This pH was chosen per theexperimentally determined pH optimum for the dextransucrase enzyme.

The fermentations consumed 450±29.3 and 468±22.3 g 40% NaOH for the A2and NC type batches, respectively.

FIG. 9 graphically illustrates the typical behavior of the pH duringfermentation after inoculation.

The growth curve (onset and log-phase) was determined by optical densityat 600 nm (OD₆₀₀), and is shown in FIG. 10.

The completion of fermentation was indicated by consumption of fructose,which was converted to mannitol, and by cessation of the take up ofalkali. Upon completion, the fermentation was harvested, and the cellsremoved via centrifugation (Sorvall RC-5B Plus, G3 rotor, 13,689 g for20 min at 5-10° C.). The broth was concentrated by evaporation (BuchiR-20 rotavap, 70° C. bath, 54° C. vapor @ 26″ Hg) to 40 brix, anddecolorized by treatment with 250 g of Carbochem CA-50S powderedactivated carbon (PAC) while still hot (50-65° C.). The slurry wasstirred for 20 minutes and vacuum-filtered (using a 2×240 mm Buchnerfunnel, 2 L side-arm flask, Whatman #3 filter papers and a 100 g Celite545 pre-coat). The powdered activated carbon cakes were washed with3×250 mL water each, and the whole wash was collected.

The minerals/salts (primarily sodium) and organic acid metabolites(primarily lactic and acetic acids) were removed in a sequentialtwo-stage ion exchange process. First, the decolorized concentrate,typically 6-7 kg at 32-36° bx, is passed through 6.8 L strong acidcation exchange resin (Purolite C-150S) to remove the minerals/salts.Then, the de-ashed broth is passed through 14.7 L weak base anionexchange resin (Purolite A-133) to remove the organic acids. Thede-ashed liquor I s then concentrated by evaporation to 56.78 brix.

The liquor (about 56 brix) is transferred hot into a 2.5 galcrystallization vessel and allowed to slowly cool to room temperature(19-22° C.) and crystallize overnight.

The resulting mixture is homogenized to yield a pourable crystal slurry.The mannitol crystals can be separated out via basket centrifuge(Robitel RA 20 VX with a 10 μm polypropylene filter bag).

A small portion of the crystal cake (0.320 kg at 95% solids, cake #1)can be washed with 500 mL ice-cold deionized water, and a 0.697 kg cakewash (wash #1) at 20.5 brix can be retained for recycle while a 2.626 kgliquor (liquor #1) at 51.70 brix can be refrigerated to 3° C. tocrystallize overnight.

Another portion of the crystals (cake #2, 0.109 kg at 95% solids) can beremoved, and washed as before. The cake wash #2 can be combined withwash #1 for recycle. The product liquor #2 at 49.0 brix can be analyzed(HPAEC-PAD, HPLC-RID, brix, pH and conductivity) and refrigeratedpending compositing with like (either A2 or Classic) batches. Oncecombined the whole composite can be evaporated to 65-67 brix prior toanalytics for the final certificate of analysis and pack out into trialdosage forms. The packaging is performed in a sterilized laminarflow-hood where 32 g amounts (for example) can be packed by volume(24.16 mL), as confirmed by lot mass, in autoclaved CRGXTA-1 oz amberglass ovals (Berry Plastics Corporation) with autoclaved 20 mm SealSafePenetrex adapter-plug seals for dose metering via luer-tip plasticsyringe (Andwin Scientific, #760020G).

Analytical Results and Reproducibility

A2 Type Product:

A2 Batch--> 021815 022315 022615 030215 033015 040915 Comp. S/M: 2.002.00 2.00 2.00 2.00 2.00 2.00 DP 1, %/bx: 2.60 1.26 1.67 1.36 0.59 1.801.51 DP 2, %/bx: 6.48 2.92 2.71 9.46 5.35 8.17 6.54 MIMO, %/bx: 74.8977.57 76.02 80.00 73.94 71.94 76.53 Mannitol, %/bx: 11.17 10.59 10.8710.03 11.08 12.11 10.42 Lactate, %/bx: 0.00 0.00 0.12 0.00 0.26 0.000.00 Formate, %/bx: 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Acetate, %/bx0.00 0.00 0.06 0.00 0.10 0.00 0.00 Glycerol, %/bx: 1.48 1.44 1.60 1.551.39 1.45 1.47 TOTAL, %/bx: 96.62 93.78 93.05 102.41 92.71 95.48 96.48PURITY: 77.51 82.71 81.70 78.12 79.75 75.35 79.33 MWD, Da: 746.89 758.02777.60 748.74 762.34 746.75 760.37 Brix, %: 50.51 49.00 53.76 54.9351.32 47.39 67.27 μS/cm: 10.53 12.8 15.65 15.19 2260 364 260 pH: 4.94.59 3.45 5.28 5.82 6.91 4.47“New Classic” or “NC”-Type Product

NC Batch--> 012015 031015 032015 032315 032515 040215 Comp S/M: 2.332.33 2.33 2.33 2.33 2.33 2.33 DP1, %/bx: 2.73 2.08 1.10 1.14 1.87 1.381.95 DP 2, %/bx: 5.54 6.96 5.93 1.96 5.47 5.06 1.73 MIMO, %/bx: 71.8871.22 73.63 74.59 69.79 76.19 72.61 Mannitol, %/bx: 13.25 10.94 10.8312.36 12.27 10.63 11.97 Lactate, %/bx: 0.000 0.000 0.000 0.000 0.9690.20 0.30 Formate, %/bx: 0.000 0.000 0.000 0.000 0.405 0.00 0.27Acetate, %/bx 0.033 0.000 0.000 0.000 0.364 0.05 0.12 Glycerol, %/bx:1.558 2.269 1.562 1.562 1.990 1.58 1.76 TOTAL, %/bx: 94.99 93.47 93.0491.61 93.13 95.10 90.72 PURITY: 75.68 76.20 79.13 81.42 74.94 80.1280.05 MWD, Da: 817.69 808.88 804.03 820.12 811.74 806.52 834.69 Brix, %:49.06 52.16 46.44 50.08 51.23 50.54 65.07 μS/cm: 239 279 43.2 350 20701415 410 pH: 6.82 6.43 6.69 7.13 6.83 6.94 5.12

Where DP 1=glucose+fructose and DP 2=sucrose+maltose:

A2 A2, %/brix 021815DA 022315DA 022615DA 030215DA 033015DA 040915DAComp. Glucose 2.27 1.11 1.46 1.26 0.56 1.69 1.41 Fructose 0.32 0.15 0.210.10 0.03 0.12 0.10 Sucrose 0.71 0.69 0.90 3.81 0.68 1.34 0.87 Maltose5.77 2.23 1.82 5.65 4.67 6.83 5.68 NC NC, %/brix 012015DA 031015DA032015DA 032315DA 032515DA 040215DA Comp Glucose 2.42 1.94 1.05 0.991.79 1.36 1.67 Fructose 0.30 0.14 0.04 0.15 0.09 0.02 0.27 Sucrose 0.751.52 1.25 0.27 1.09 0.70 0.36 Maltose 4.79 5.44 4.68 1.69 4.38 4.36 1.37

Comparative chromatograms (HPAEC-PAD) and 300 MHz 1H NMR for the twocomposite products are provided in FIGS. 11 through 18.

The MIMO of these products had a reproducible distribution of DPs from3-9 that varied according to S/M present at the start of fermentation:

A2, RSD, NC, RSD, N = 6 each mean: STDEV: %: mean: STDEV: %: MIMO-DP312.522 1.003 8.007 8.88 0.50 5.61 MIMO-DP4 23.510 1.173 4.991 17.46 1.005.75 MIMO-DP5 23.357 0.685 2.931 22.59 1.38 6.12 MIMO-DP6 10.888 0.4233.883 13.73 0.28 2.06 MIMO-DP7 3.280 0.181 5.515 5.09 0.38 7.52 MIMO-DP82.251 0.229 10.167 3.96 0.41 10.35 MIMO-DP9 0.683 0.047 6.933 1.14 0.1714.53

Example 2

After performing the processes described in Example #1, it was observedthat mannitol would crystallize from the final products uponrefrigeration or long-term storage at cooler room temperature. Thisexample illustrates the variability in product composition relative tothe crystallization process employed. Additionally, the batch generatedas described in this Example was used to test the effect ofsterilization (or steam) in place on sucrose:maltose ratio.

Improved Composition Via Manipulation of Crystallization Parameters

To a 20 L fermenter (New Brunswick BioFlo 410) was added the followingmedium:

Batch: 051315 kg: g: Water 12.600 Sucrose 1.800 Maltose-H₂O 0.998MnSO₄—H₂O 0.00015 0.15083 MgSO₄ 0.00146 1.46045 FeSO₄—7H₂O 0.000150.14997 KH₂PO₄ 0.04000 40.00375 NaCl 0.00015 0.14985 CaCl₂—2H₂O 0.000800.80170 Yeast Extract 0.077 NaOH, 50% 0.017 16.68234 Total: 15.534 TS,%: 17.374 Brix, %: 18.198 S/M: 2.00

The pH of the medium was adjusted to 7.00 with NaOH (50%) and thesterilized medium (pre-inoculation) was sampled for analysis of S/M viaHPLC-RID. 200 mL of the medium was transferred to an Erlenmeyer flask,the flask was then sealed and autoclaved at 121° C. for 15 minutes. Themedium was cooled, and inoculated with 1 mL vial stock (0.5 mL late-logculture Leuconostoc citreum ATCC 13146, NRRL B-742, in 20% glycerol,stored at −75° C.). The seed was incubated with swirling at 27° C. andallowed to grow for 16 Hr.

The balance of the medium was transferred to the fermenter via sanitarypump. The fermenter was sealed and sterilized in place (SIP) to 116° C.,as previously described in Example 1. The fermenter was inoculated with150 mL of the flask seed culture and the pH of the medium was correctedto 6.50 using 37% HCl. Fermentation was allowed to proceed for 55 Hrwith pH adjustment with 40% NaOH (40%) to maintain 5.50.

Typically, there is a 6 hour induction period preceding log-growth.Log-growth typically proceeded for approximately 10 hours. A pH 5.5 wastypically achieved within about 2 hours of log-phase growth, and this pHis maintained thereafter.

The fermentation consumed 464 g of 40% NaOH.

The completion of fermentation was indicated by consumption of fructose,which was converted to mannitol, and by cessation of the take up ofalkali. The fermentation was harvested, and the cells removed viacentrifugation (Sorvall RC-5B Plus, G3 rotor, 13,689 g for 20 min at5-10° C.). The broth was concentrated by evaporation (Buchi R-20rotavap, 70° C. bath, 54° C. vapor @ 26″ Hg) to 40 brix, and decolorizedby treatment with 266 g of powdered activated carbon (PAC CarbochemCA-50S) while still hot (50-65° C.). The slurry was stirred for 20minutes and vacuum-filtered (2×240 mm Buchner funnel, 2 L side-armflask, Whatman #3 filter papers and a 100 g Celite 545 pre-coat). ThePAC cakes were washed with 3×250 mL water each, and the whole collected.

The minerals/salts (primarily sodium) and organic acid metabolites(primarily lactic and acetic acids) were removed in a sequentialtwo-stage ion exchange process. First 6.236 kg of broth at 35.5 brix waspassed through 6.8 L strong acid cation exchange resin (Purolite C-150S)to remove the minerals/salts. Then, the de-ashed broth is passed through14.7 L weak base anion (Purolite A-133) to remove the organic acids. ThepH of the de-ashed liquor was adjusted to 6.16 (from 10.80) with HCl(37%), and concentrated by evaporation to 57.01 brix. The de-ashedconcentrate was transferred hot into a 2.5 gal crystallization vesseland allowed to slowly cool to room temperature (19-22° C.) andcrystallize overnight.

The resulting mixture was homogenized to yield a pourable crystalslurry. The mannitol crystals were separated via basket centrifuge(Robitel RA 20 VX with a 10 μm polypropylene filter bag).

A small portion of the crystal cake (0.279 kg at 95% solids, cake #1)was washed with 500 mL ice-cold deionized water, and a 0.672 kg cakewash (wash #1) at 18.7 brix was retained for recycle while 2.403 kg ofliquor (liquor #1) at 52.56 brix was collected.

Liquor #1 was concentrated by evaporation to 65.68 brix. The resultingconcentrate was split into two portions. The first portion (0.921 kg)was crystallized again at room temperature (19-22° C.). The secondportion was crystallized to a temperature of approximately 2° C. Bothcrystallizations were allowed to proceed overnight.

The room temperature crystals (cake #2RT, 0.039 kg at 95% solids) wereremoved, as described above. The cake wash #2RT (0.552 kg) was combinedwith wash #1 for recycle.

The refrigerated crystals (cake #2RC, 0.094 kg at 95% solids) wereremoved, as before. The cake wash #2RC (0.626 kg) was combined with wash#1 for recycle.

The product liquors #2RT at 64.08 brix and #2RC at 63.82 brix wereanalyzed via (HPAEC-PAD, HPLC-RID, brix, pH and conductivity).

Results

HPLC-RID determined that the pre-inoculated sucrose/maltose ratio hadincreased from 2.00 to 2.73 after sterilization in process:

%/brix %/brix Compound: Pre SIP Post SIP Sucrose 62.26 61.14 Maltose31.31 22.38 Glucose 0.27 4.61 fructose 0.00 0.15 Total: 93.84 88.28 S/M:1.99 2.73

The composition of the products produced as described in this Exampleafter cold and room temperature second crystallization are shown below:

Room Cold Temp 2nd Xl, 2nd Xl, T° C.: 2%/ T° C.: 20%/ Component brix: %w/w: Component brix: % w/w: Glucose 0.79 0.50 Glucose 0.76 0.49 Fructose0.03 0.02 Fructose 0.02 0.02 Sucrose 1.76 1.12 Sucrose 1.62 1.04 Maltose4.93 3.15 Maltose 4.84 3.10 MIMO 89.08 56.85 MIMO 87.75 56.23 Mannitol8.82 5.63 Mannitol 11.20 7.18 Lactate 0.33 0.21 Lactate 0.32 0.20Glycerol 0.38 0.24 Glycerol 0.37 0.24 Formate 0.00 0.00 Formate 0.000.00 Acetate 0.13 0.09 Acetate 0.11 0.07 TOTAL: 106.25 67.81 TOTAL:106.99 68.56 Brix, %: 63.82 Brix, %: 64.08 Purity, %: 83.84 Purity, %:82.02 Mw, Da: 737 Mw, Da: 739 MIMO DP %/brix: MIMO DP %/brix: MIMO-DP313.84 MIMO-DP3 13.71 MIMO-DP4 31.62 MIMO-DP4 30.82 MIMO-DP5 27.75MIMO-DP5 27.30 MIMO-DP6 11.50 MIMO-DP6 11.48 MIMO-DP7 3.11 MIMO-DP7 3.19MIMO-DP8 1.26 MIMO-DP8 1.25 MIMO-DP9 0.00 MIMO-DP9 0.00

Thus, for batches made via cold filter sterilization of the sugars, asucrose/maltose ratio of 2.73 should give a similar MWD, e.g. 740 to 790Da.

Increasing the brix of liquor #1 to greater than 65 and cooling thesecond crystallization to 2-5° C. improved the purity of the product(relative to room temperature crystallization of either 52 or 65.78brix) by approximately 26% (−3.15% Thrix). The final product so obtaineddemonstrated improved shelf stability and did not crystallize furtheronce stored at either 5 or 20° C.

This process-iteration (concentration of liquor #1 and sequentialcrystallization, first to room temperature and then to 2-5° C.) and thecomposition obtained thereby have been integrated into thecommercial-scale process.

Example 3

This Example demonstrates the fermentation at 10 L scale, using asucrose:maltose ratio of 2.00 at time of inoculation, and withintroduction of sugars via filtration through a 0.2 mm filter(sterilized by filtration, SBF).

10 L Trial Fermentation at S/M=2.0 with SBF of Sugars

A sugar and salt stock solution was prepared to contain the following:

10 L #1 kg: Water 4.471 Sucrose 1.430 Maltose-H₂O 0.795 NaCl 0.00012CaCl₂—2H₂O 0.00064 Total: 6.698 TS, %: 32.05 Brix, %: 32.06 S/M: 1.990The following components were added to a 10 L fermenter (BioFlo 410 orequivalent):

SIP 10 L #1 kg: Water 4.500 MnSO₄—H₂O 0.000098 MgSO₄ 0.000951 FeSO₄—7H₂O0.000098 KH₂PO₄ 0.02606 Yeast Extract 0.04887 Total: 4.576 TS, %: 0.00Brix, %: 1.66 S/M: n/aThis mixture was sterilized in place (within the fermenter) at 121° C.for 30 minutes then cooled to room temperature, and 5.4697 kg of thesugar and salt stock solution was transferred (SBF) into the fermentervia 0.2 μm filter to give a pre-inoculation (sampled for analysis)medium with the following composition:

Total 10 L #1 kg: Water 8.402 Sucrose 1.168 Maltose-H₂O 0.650 MnSO₄—H₂O0.000 MgSO₄ 0.001 FeSO₄—7H₂O 0.000 KH₂PO₄ 0.026 NaCl 0.000 CaCl₂—2H₂O0.001 Yeast Extract 0.049 NaOH, 50% 0.000 Total: 10.296 TS, %: 17.03Brix, %: 17.77 S/M: 1.990The medium was adjusted to pH 6.50 with 50% NaOH, and inoculated with100 g of late-log L. citreum NRRL B-742 grown in medium of the samecomposition. The fermentation was allowed to proceed for 55 hours withpH control to maintain the pH at 5.5 once that pH was achieved viabacterial acidogenesis. The fermenter was sampled at regular intervalsfor analysis via HPAEC-PAD and HPLC-RID.Results:

The sucrose:maltose (S/M) ratio of the pre-inoculation medium wasconfirmed by HPLC-RID (BioRad Aminex, HPX-87P, 80° C., water at 0.6mL/min):

-   -   %/brix

%/brix Compound: pre inoc: Sucrose 61.88 Maltose 32.00 Glucose 0.25fructose 0.00 Total: 94.14 S/M: 1.93

The amount of feedstock (sucrose and maltose), product (MIMO),intermediate (fructose), and byproducts (total organic acids andmannitol) over time as detected via HPLC-RID and HPAEC-PAD, are given inFIG. 19. The evolution of the mass-average molecular weight of the MIMOis shown in FIG. 20.

Ultimately, the starting sucrose:maltose ratio of about 2.00 (1.93 atthe time of inoculation) yielded 51.17%/brix MIMO (60.11%/total sugars)that had a mass-average MWD of 642.46. See chart shown in FIG. 40.

Example 4

This Example illustrates that fermentation at the 10 L scale, using asucrose:maltose ratio of 2.75 at time of inoculation, with introductionof sugars via filtration through a 0.2 μm filter (sterilized byfiltration, SBF), will give a MWD similar to that which arises from afermentation batch with a starting sucrose:maltose ratio of 2.00 priorto SIP (i.e. approx. 2.73 at time of inoculation, see FIG. 8).

10 L Trial Fermentation at S/M=2.75 at the Time of Inoculation with SBFof Sugars

Experimentally, this fermentation is identical to that demonstrated inExample #3 with the exception that the amount of both sucrose andmaltose have been altered in order to achieve a S/M of 2.75 at the timeof inoculation whilst maintaining a total sugar of approximately 17-18%w/w.

The final sterile medium contained:

Total 10 L #2 kg: Water 8.740 Sucrose 1.333 Maltose-H₂O 0.536 MnSO₄—H₂O0.000 MgSO₄ 0.001 FeSO₄—7H₂O 0.000 KH₂PO₄ 0.026 NaCl 0.000 CaCl₂—2H₂O0.001 Yeast Extract 0.049 NaOH, 50% 0.000 Total: 10.685 TS, %: 16.99Brix, %: 17.70 S/M: 2.751

The fermentation behaved normally for L. citreum NRRL B-742 grown onthis medium and with pH controlled at 5.5 using 50% NaOH. The amounts offeedstock (sucrose and maltose), product (MIMO), intermediate(fructose), and byproducts (total organic acids and mannitol) over timeas detected by HPLC-RID and HPAEC-PAD, are given in FIG. 21. Note thatthe metabolic activities and MIMO yield are essentially the same asthose shown in Example #3 (compare FIG. 21 to FIG. 20), but theevolution of the mass-average molecular weight of the MIMO, shown inFIG. 22, is quite different and reflects the increased sucrose:maltoseratio.

Ultimately, fermentation at a sucrose:maltose ratio of about 2.75(2.72-2.75) at the time of inoculation yielded 53.32%/brix MIMO(57.44%/total sugars) with a mass-average MWD of 760.73 Da. This isconsistent with results shown in Example #2 where the pre-inoculatedsucrose/maltose ratio had increased from 2.00 to 2.73 duringsterilization in place (SIP).

FIG. 41 shows the composition of the fermentation broth so obtained atthe left, with the distribution of MIMO at the right.

Example 5

Colored by-products remained in the final product (imparting a browncolor and caramel-like flavor) when using the processes described abovein Examples 1, 2, and 3. While not undesirable (organoleptically), it isa difficult parameter to control, and it was preferable to avoiddestroying maltose from a cost perspective (it is expensive). With theseconsiderations in mind scale-up experiments were designed to test SIP ofbulk water and minerals, and filter sterilization of the carbohydratecomponents. As illustrated in this Example, such a process avoided colorformation and facilitated more precise control over the S/M (fixed at2.75).

Improved Composition for Scale-Up

To a 2 L fermenter (New Brunswick Celligen 512) was added:

Batch: 51815 kg: g: Water 1.752 MnSO₄—H₂O 0.00002 0.02093 MgSO₄ 0.000200.20312 FeSO₄—7H₂O 0.00002 0.02160 KH₂PO₄ 0.00557 5.56501 NaCl 0.000020.02068 CaCl₂—2H₂O 0.00011 0.11113 Yeast Extract 0.01043 10.42705 NaOH,50% 0.00096 0.95946 Total: 1.769This mixture was sealed in the fermenter and autoclaved at 121° C. for15 minutes. While the contents of the fermenter were still hot (80-90°C.), 0.270 kg of sucrose and 0.108 kg of maltose monohydrate weretransferred into the fermenter and dissolved via strong agitation at 400RPM. Once cooled to 27° C., the whole mixture was sampled for analysisvia HPLC-RID, confirming that the sucrose:maltose ratio was 2.72.

The fermenter was inoculated with 20 mL late-log L. citreum NRRL B-742,made as previously described, and the pH was controlled as previouslymentioned. Fermentation was allowed to proceed for 62 Hr with dailysampling. During this time, the fermentation consumed approximately 43 gof 40% NaOH.

At 62 Hr, the whole batch was harvested and the cells removed, aspreviously described, to give 17.8 brix broth with a conductivity of19.1 mS/cm. The broth was of much lower color, e.g. 1039.5 IU relativeto 11,799 IU for the same (complete) media run through and SIP cycle.Due to the reduced color, the requirement for powdered activated carbonwas reduced by a factor of four, and still have headroom within a factorof two.

The broth was concentrated to 45.0 brix by evaporation, and decolorizedwith 0.1333% (over starting mass of medium) CA-50S PAC (29.2 g), aspreviously described.

The minerals/salts and organic acids were removed from 1.075 kg ofdecolorized liquor at 39 brix as previously described. The combinedde-ashed liquor was adjusted to pH 6.16 (from pH 10.80) with 37% HCl(8.73809 g) and the whole de-ashed liquor concentrated by evaporation to57.04 brix.

The de-ashed concentrate was transferred hot into a one litercrystallization vessel and allowed to slowly cool to room temperature(19-22° C.) and crystallize overnight.

The resulting mixture was homogenized to yield a pourable crystalslurry. The mannitol crystals were separated via basket centrifuge(Robitel RA 20 VX with a 10 μm polypropylene filter bag). The crystalcake (0.320 kg at 95% solids, cake #1) was washed, in small portions,with 500 mL ice-cold deionized water. 0.697 kg cake washings (wash #1)at 20.5 brix were retained for recycle. 2.626 kg liquor (liquor #1) at51.70 brix was refrigerated to 3° C. and allowed to crystallizeovernight.

The crystals (cake #2, 0.109 kg at 95% solids) were removed, as before.The cake wash #2 was combined with wash #1 for recycle. The productliquor #2 at 49.0 brix was analyzed (HPAEC-PAD, HPLC-RID, brix, pH andconductivity).

Results

The composite results via HPAEC-PAD and HPLC-RID are given below.

Hr: 15 39 63 brix: 18.2 17.8 17.8 mannitol 25.88 26.50 26.49 glucose0.02 0.13 0.67 fructose 1.73 0.06 0.01 sucrose 0.22 0.13 0.23 maltose2.35 2.49 3.01 DP 3 10.71 7.41 7.39 DP 4 20.26 16.83 13.98 DP 5 17.4916.90 15.68 DP 6 6.29 7.56 7.87 DP 7 1.58 2.16 2.49 DP 8 0.79 0.91 1.21DP 9 0.00 0.00 0.00 lactate 9.55 13.01 13.17 glycerol 0.00 0.00 0.00formate 0.00 0.00 0.00 acetate 4.52 4.77 4.85 TOTAL: 102.02 100.51 98.77MIMO, %: 57.13 51.76 48.62 Purity, %: 56.00 51.50 49.22 MWD: 727.43754.80 761.03 Yield %: 57.78 51.97 49.68

Example 6

The process incarnations described in the previous examples (1-5) wereintegrated and scaled up to 3000 L with a theoretical overall processyield of 240 kg MIMO (DS)/total sugars fed. These examples detail thescaled process, and the composition obtained thereby.

Commercial-Scale Production of MIMO

Fermentation/MIMO Biosynthesis

To 3.7 kg RO (reverse osmosis) water was added sucrose (refined white,from cane), 0.5302 kg; maltose monohydrate (Sunmalt-S[N]), 0.2935 kg;yeast extract (Marcor bacteriological grade), 0.0221 kg; potassiumphosphate monobasic, 0.0118 kg; magnesium sulfate (anhydrous), 0.00043kg; ferrous sulfate heptahydrate, 0.000045 kg; manganese sulfatemonohydrate, 0.000045 kg, sodium chloride, 0.000045 kg, and calciumchloride dihydrate (USP), 0.00024 kg.

The pH of the medium was adjusted to 7.0 with 50% NaOH (FCC), 0.0057 kg.

700 mL of medium was dispensed into each of six unbaffled Fernbachflasks. The flasks were sealed using foam plugs and autoclaved at 121°C. for 15 minutes.

Five of the six flasks were inoculated with 1 mL each of vial stock(Leuconostoc citreum NRRL B-742; 0.5 mL late-log culture+0.5 mLglycerol, 40%, certified Kosher-Pareve). The sixth flask was anuninoculated control.

The flasks were incubated at 27° C. for 16 Hr (OD₆₀₀=1.476±0.03) withagitation at 150 RPM. The inoculum was inspected via microscopy todetermine the culture was clean prior to use.

The following was added to a batch tank: water purified by reverseosmosis (RO water) 1440 kg; sucrose, 498.96 kg; maltose monohydrate, 200kg; sodium chloride, 0.037 kg; and calcium chloride dihydrate, 0.204 kg.

In the meantime, a 1200 gallon seed fermenter was cleaned in place. Tothe seed fermenter was added RO water, 238 kg; yeast extract, 2.76 kg;potassium phosphate monobasic, 1.48 kg; magnesium sulfate (anhydrous);0.054 kg, ferrous sulfate heptahydrate, 0.0057 kg; and manganese sulfatemonohydrate, 0.0057 kg.

The contents of the fermenter were thoroughly mixed, allowed to rest at37° C. for two hours, and then sterilized in place at 121° C. for 60minutes.

Once cooled, 309.2 kg of the sugar and salt solution was transferredfrom the charge tank to the seed fermenter through a sterilizing 0.2 μmfilter capsule with a 1.0 μm pre-filter (20′ Cuno cartridge filter). Thefilter and lines were washed through with 10 kg of RO water. The mixedmedium had a pH of 5.47.

The seed fermenter was inoculated with 3.8 kg late-log flask culture.The fermentation was allowed to proceed under 1-3 psig air (in headspaceto maintain positive pressure), at 27° C., with agitation at 42 RPM for16 hours (OD₆₀₀=2.74).

In the meantime, into a cleaned in place production fermenter was addedRO water, 1332 kg, yeast extract, 15.01 kg; potassium phosphatemonobasic, 8.01 kg; magnesium sulfate (anhydrous), 0.2922 kg; ferroussulfate heptahydrate, 0.030 kg, and manganese sulfate monohydrate, 0.030kg. This mixture was sterilized in place at 121° C. for 60 mins. Intothis mixture was pumped 1141 kg of the sugar and salt solution (from thecharge tank) through a sterilizing 0.2 μm filter capsule with a 1.0 μmpre-filter (20′ Cuno cartridge filter).

Thirty-one kg of late-log seed culture (L. citreum NRRL B-742.) was usedto inoculate the production fermenter. The pH was adjusted to 6.52 with50% NaOH, and the fermentation was allowed to proceed for 55 hours at27° C., 1-3 psig air, and with agitation at 31 RPM. The pH wasmaintained at 5.5 with 50% NaOH (appx 120 kg).

FIG. 23 graphically illustrates the pH (3.32 final) and the OD₆₀₀ (2.74final) in the seed tank, while FIGS. 24-26 demonstrate the pH (5.58final), the OD₆₀₀ (8.78 final) in the production fermenter, thecomposition of the broth therein made, and MIMO molecular weight overtime during the fermentation.

Downstream Processing/Work Up

The biomass (cells, etc.) was removed from the fermentation broth viapassage through a 0.2 μm microfilter (skid). Any remaining MIMO held upin the retentate was recovered via six stages of diafiltration. Thepermeate and diafiltrate were combined and evaporated to approximately40 brix via wiped film evaporator (WFE). The resulting concentrate wasdischarged hot and treated with 12.5 kg of powdered activated carbon(PAC, Carbochem CA-50S) and 21 kg Celite 545 diatomite filter aid. Thewhole mixture was stirred for 20 minutes before filtration through afilter press with a 20 kg Celite 545 pre-coat. A 1 μm cartridge filterwas used to polish fines from the filtrate. RO water, 700 kg was used towash the PAC cake.

The filtrate and PAC wash were combined to give 1277 kg of decolorizedconcentrate at 29.3 brix.

The decolorized concentrate was de-ashed via passage (5×300 kg slugs)through strong acid cation (SAC, Purolite C-150S, H⁺ form, 14.0 cuft)and a weak base anion (WBA, Purolite A-133, free-base form, 13.5 cuft)ion exchange resins.

The combined ion exchange (IEX) product (5.6 brix) was filtered througha 0.2 mm capsule filter into a cleaned in place holding tank where itwas adjusted to pH<4.2 (2.8, actual) with 31% hydrochloric acid.

The acidified IEX product was concentrated to 54.21 brix via evaporation(WFE), discharged hot into 2×1 m³ stainless steel totes. These wereallowed to slowly cool, with slow agitation (pneumatic mixer) to roomtemperature (25° C.).

The crystals were removed from the mother liquor via passage through aHastalloy nutsch filter (10 mm filter disk and Celite 545 pre-coat) toyield 273 kg of liquor #1.

The crystal cake (153 kg) was washed with cold RO water, 285 kg to yield342 kg cake wash #1 and 60 kg cake #1.

Cake wash #1 was frozen for recycle into a future batch and Liquor #1was evaporated to 65.60 brix by evaporation (pot still). The resultingliquor was discharged hot into a 1 m³ stainless steel tote and allowedto slowly cool, with slow agitation (pneumatic mixer) to roomtemperature (25-30° C.). Then, the tote was moved into a freezer wherethe crystallization was continued with slow cooling to 5° C.

The crystals were removed from the mother liquor via passage through aHastalloy nutsch filter (10 mm filter disk and Celite 545 pre-coat) toyield 127.1 kg of liquor #2 at 64.04° brix. The crystal cake was washedwith cold RO water to yield 184 kg cake wash #2 and 39.4 kg cake #2.

Cake wash #2 was frozen for storage and recycle into the next batch.

Liquor #2 was pasteurized at 70° C. for 30 minutes in the pot still,cooled and packaged into 55 gallon sanitized poly drums.

Samples were submitted for microbiological testing and to analytics forissuance of batch COA.

The composition thereby made was as shown in FIG. 42 (as detected byHPAEC-PAD/HPLC-RID).

FIG. 27 shows a HPAEC-PAD chromatogram of product lot #150622 whereinthe components are identified as A: D-mannitol; B: L-arabinose (internalstandard); C: D-glucose; D: D-fructose; E: sucrose; F: maltose; andwhere G-M correspond to MIMO DP 3-9.

FIG. 28 shows HPAEC-PAD chromatograms during removal of mannitol frommother liquor 1, and 2, 3 corresponding to compound crystallizationstages, 1 and 2, respectively. The components are identified as A:D-mannitol; B: L-arabinose (internal standard); C: D-glucose; D:D-fructose; E: sucrose; F: maltose; and G-M corresponding to MIMO DP3-9. Note that mannitol is reduced and that the MIMO purity increasedthereby.

FIG. 29 shows an overlay of 1D 300 MHz ¹H NMR (water suppressed in D₂O)spectra of the anomeric regions of 1 and 2, NC and lot #150622 products,respectively and, 3, D-panose (δ reference) wherein signal A correspondto α-(1,4) anomeric protons; signal B corresponds to α-(1,6) anomericprotons; signals C and D correspond to the reducing α and β anomericprotons, respectively, at the reducing end; and signals E and Fcorrespond to regions corresponding to α-(1,3) and α-(1,2) anomericprotons, respectively. Note the minimal or absent second doublet in lot#150622; this is likely due to the acidification step which avoidspossible alkali catalyzed epimerization of C2.

FIG. 30 shows an overlay of 1D 300 MHz ¹H NMR (water suppressed in D₂O)spectra of the non-anomeric regions of 1, NC and, 2, lot #150622products.

FIG. 31 shows an overlay of 1D 300 MHz ¹H NMR (water suppressed in D₂O)spectra of the alkyl regions of (1) lot #150622; and (2) A2 products.Signal A is an unknown; signal B corresponds to acetyl protons (thedifference is due to pH, A2=4.81, lot #150622=6.08); signal Ccorresponds to lactate C3 methyl protons; and signal D is unassigned.Note, the amount of acetate/lactate in both 1 and 2 (A2) were below theminimum detectable limit (MDL) of HPLC-RID.

Example 7

The composition and process described in Example 6 was modified toincrease product recovery (minimizing losses and materials retained forbatch-recycle). The modified process and composition so produced aredetailed here.

Modified Process and Composition Made Thereby—the State of the Art

Fermentation/MIMO Biosynthesis

To 3.7 kg RO (reverse osmosis) water was added sucrose (refined white,from cane), 0.5302 kg; maltose monohydrate (Sunmalt-S[N]), 0.2935 kg;yeast extract (Marcor bacteriological grade), 0.0221 kg; potassiumphosphate monobasic, 0.0118 kg; magnesium sulfate (anhydrous), 0.00043kg; ferrous sulfate heptahydrate, 0.000045 kg; manganese sulfatemonohydrate, 0.000045 kg, sodium chloride, 0.000045 kg, and calciumchloride dihydrate (USP), 0.00024 kg.

The pH of the medium was adjusted to 7.0 with 50% NaOH (FCC), 0.0057 kg.

700 mL of medium was dispensed into each of six unbaffled Fernbachflasks. The flasks were sealed using foam plugs and autoclaved at 121°C. for 15 minutes.

Five of the six flasks were inoculated with 1 mL each of vial stock(Leuconostoc citreum NRRL B-742; 0.5 mL late-log culture+0.5 mLglycerol, 40%, certified Kosher-Pareve). The sixth flask was anon-inoculated control.

The flasks were incubated at 27° C. for 16 Hr (OD₆₀₀=1.476±0.03) withagitation at 150 RPM. The inoculum was inspected via microscopy todetermine the culture was clean prior to use. A sample was taken andfrozen at −75° C. in 20% w/w glycerol for later analysis via 16S rRNAsequencing to determine and verify culture purity.

In the meantime, a 1200 gallon seed fermenter was cleaned in place.

A batch tank was loaded with RO water, 1510 kg; sucrose, 522.04 kg;maltose monohydrate, 210 kg; sodium chloride, 0.039 kg; and calciumchloride dihydrate, 0.214 kg.

To the seed fermenter was added RO water, 238 kg; yeast extract, 2.80kg; potassium phosphate monobasic, 1.50 kg; magnesium sulfate(anhydrous) 0.055 kg; ferrous sulfate heptahydrate, 0.0057 kg; andmanganese sulfate monohydrate, 0.0059 kg.

The contents of the fermenter were thoroughly mixed, allowed to rest at37° C. for two hours, and then sterilized in place at 121° C. for 60minutes.

Once cooled, 310 kg of the sugar and salt solution was transferred fromthe charge tank to the seed fermenter through a sterilizing 0.2 μmfilter capsule with a 1.0 μm pre-filter (20′ Cuno cartridge filter). Thefilter and lines were washed through with 10 kg of RO water. The mixedmedium had a pH of 5.54.

The seed fermenter was inoculated with 3.8 kg late-log flask culture.The fermentation was allowed to proceed under 1-3 psig air (in headspaceto maintain positive pressure) at 27° C., with agitation at 42 RPM for16 hours (OD₆₀₀=2.805, pH 3.45).

In the meantime, the following were added to a cleaned in placeproduction fermenter: RO water, 1332 kg; yeast extract; 5.6 kg;potassium phosphate monobasic, 8.05 kg; magnesium sulfate (anhydrous),0.2922 kg; ferrous sulfate heptahydrate, 0.030 kg; and manganese sulfatemonohydrate, 0.030 kg. The fermentation contents were sterilized inplace at 121° C. for 60 mins. The following was pumped into theproduction fermenter: 1680 kg of the sugar and salt solution (from thecharge tank) through a sterilizing 0.2 μm filter capsule with a 1.0 μmpre-filter (20′ Cuno cartridge filter). The filter and lines were washedthrough with 10 kg of RO water.

Thirty-one kg of late-log seed culture was used to inoculate theproduction fermenter. The pH was adjusted to 6.54 with 50% NaOH (˜1 kg),and the fermentation was allowed to proceed for 55 hours at 27° C., with1-3 psig air, and with agitation at 31 RPM. The pH was maintained at 5.5with 50% NaOH (appx 120 kg). A sample was taken and frozen at −75° C. in20% w/w glycerol for later analysis via 16S rRNA sequencing to determineand verify culture purity.

FIG. 32 shows the amounts of chemical species as detected by HPAEC-PADand HPLC-RID throughout the course of a 3000 L fermentation (S/M=2.75,lot #151105) using L. citreum NRRL B-742.

FIG. 33 illustrates the evolution of the molecular weight distributionof MIMOs throughout the course of a 3000 L fermentation (S/M=2.75, lot#151105) with L. citreum NRRL B-742. Note that the MWD continues toincrease (until about 15 hours) after the sucrose is exhausted (at about10 hours). The rate of chain growth then takes place at a lower, butconstant rate until the end of fermentation (55 hours) when themolecular weight of about 776.5 Da was achieved.

Downstream Processing/Work Up:

The biomass (cells, etc.) was removed from the fermentation broth viapassage through a 0.2 μm microfilter (skid). Remaining MIMO held up inthe retentate was recovered via six stages of diafiltration. Thepermeate and diafiltrate were combined (6745 kg at 8.6° brix) andevaporated to 48.31 brix using a wiped film evaporator (WFE). The WFEwas washed with RO water and the washings retained for further use (WFEwash #1).

The resulting concentrate was discharged hot and treated with 12.5 kg ofpowdered activated carbon (PAC, Carbochem CA-50S) and 21 kg Celite 545diatomite filter aid. The mixture was stirred for 20 minutes beforefiltration through a filter press with a 20 kg Celite 545 pre-coat. A 1μM cartridge filter was used to polish fines from the filtrate. Washwater (11.13 brix out) from the wiped film evaporator (WFE wash #1, 700kg), followed by an RO water push (1.7 brix out), was used to wash thePAC cake.

The filtrate and PAC wash were combined to give 1873 kg of decolorizedconcentrate at 25.7 brix. The decolorized concentrate was de-ashed viapassage (5×325 kg slugs) through strong acid cation (SAC, PuroliteC-150S, H⁺ form, 14 cuft) and a weak base anion (WBA, Purolite A-133,free-base form, 13.5 cft) ion exchange resins.

The combined ion exchange product (5.6 brix) was filtered through a 0.2μm capsule filter (30″ PRMXE) into a cleaned in place holding tank whereit was adjusted to pH<4.2 (2.8, actual) with 85% phosphoric acid.

The acidified ion exchange product was concentrated to 52.64 brix viaevaporation using a wiped film evaporator, and then discharged hot into2×1 m³ stainless steel totes. These were allowed to slowly cool, withslow agitation (pneumatic mixer) to room temperature (25° C.). The wipedfilm evaporator was rinsed with RO water and retained for further usedownstream in the process (1.7 brix, WFE wash #2).

The crystals were removed from the mother liquor via passage through aHastalloy nutsch filter (10 mm filter disk and Celite 545 pre-coat) toyield 571 kg of liquor #1 at 49.7° brix.

The 118 kg crystal cake was washed with cold wiped film evaporator rinsewater (WFE wash #2) to yield 218 kg cake wash #1 at 24.3 brix and 54.4kg cake #1.

Liquor #1, cake wash #1, and any remaining WFE wash #2 were combined,and evaporated to 67.7 brix by evaporation (pot still). The resultingliquor was cooled to 30° C. and discharged into a 1 m³ stainless steeltote. Then, the tote was moved into a freezer where the crystallizationwas continued with slow cooling to 5° C. The pot still was rinsed with200 kg RO water to yield 196 kg wash at 1.2° brix (still wash) which wasrefrigerated for downstream use.

The crystals were removed from the mother liquor via passage through aHastalloy nutsch filter (10 mm filter disk and Celite 545 pre-coat) toyield 315 kg of liquor #2 at 63.5° brix.

The 165.25 kg crystal cake was washed with the cold still wash to yield248 kg cake wash #2 at 27° brix and 77.65 kg cake #2.

Cake wash #2 and any remaining pot-still rinse was frozen for storageand recycle into the next batch.

315 kg Liquor #2 was pasteurized at 70° C. for 30 minutes in a potstill, cooled and packaged into 55 gallon Scholle bags (Bag in box) withsanitary fittings. The yield was 314.3 kg pasteurized product containing170.3 kg MIMO at 63.01 brix.

Samples were submitted for microbiological testing and to analytics forissuance of batch certificate of analysis. Chromatograms comparing lot#150622 with this one, lot #151105, are overlaid and given in FIG. 34.

Fermentation yield was 51.23% over total sugars fed. Process recoverywas, overall, 62.40% with 77.52% potentially recoverable or 31.97% yieldover total sugars fed. The overall balance of mass for the DSP was(significant points in bold-text):

Purity, ISOT, Sample: kg: Brix: DS, kg: %: kg: Final broth 3081 18.10557.63 48.94 272.92 permeate init 2175 18.00 391.50 46.82 183.30retentate init 2250 18.00 405.00 49.28 199.58 Perm Stage 1 825 10.183.33 46.82 39.01 Perm Stage 2 750 6.1 45.75 46.82 21.42 Perm Stage 3750 3.3 24.75 46.82 11.59 Perm Stage 4 750 2.0 15.00 46.82 7.02 PermStage 5 750 1.3 9.75 46.82 4.57 Perm Stage 6 750 0.7 5.25 46.82 2.46Pooled permeate 6745 8.6 580.07 46.82 271.59 Retentate Stage 1 750 10.377.25 49.28 38.07 Retentate Stage 2 750 6.1 45.75 49.28 22.54 RetentateStage 3 750 3.5 26.25 49.28 12.94 Retentate Stage 4 750 2.0 15.00 49.287.39 Retentate Stage 5 750 1.3 9.75 49.28 4.80 Retentate Stage 6 750 0.96.75 49.28 3.33 WFE concentrate tote #1 781 47.9 374.10 48.88 182.87 WFEconcentrate tote #2 329 49.3 162.20 48.88 79.29 Pooled WFE concentrate1110 48.31 536.30 51.41 262.16 WFE distillate 5544 0.2 11.09 51.41 5.70WFE wash 1 700 1.8 12.60 51.41 6.48 Product filtrate tote #1 756 32241.92 48.88 118.26 Product filtrate tote #2 243 40 97.20 48.88 47.51Filter press wash 634 11.3 71.64 50.36 36.08 RO wash 2400 1.7 5.10 53.822.74 Pooled Product Filtrate 1873 25.70 481.36 50.86 244.83 Pandrippings 198 15.20 30.10 50.47 15.19 IEX pulse #1 1317 5.0 66.18 57.6538.16 IEX pulse #2 1370 5.4 73.98 57.65 42.65 IEX pulse #3 1619 5.182.57 57.65 47.60 IEX pulse #4 1749 4.9 85.70 57.65 49.41 IEX pulse #51855 4.7 87.19 57.65 50.27 IEX pulse #6 1629 1.8 29.45 57.65 16.98Pooled IEX effluent 9426 4.51 425.06 57.65 245.07 0.2 mm filtration RTn/a acidification 0.5 n/a WFE IEX concentrate 695 58.68 407.83 57.65235.13 WFE IEX distillate 6835 0.2 13.67 57.65 7.9 WFE IEX wash #1 2501.7 4.25 57.65 2.5 WFE IEX wash #2 250 0.1 0.25 57.65 0.1 Stage 1 liquor571 49.7 283.79 71.62 203.2 Stage 1 crystals 153.7 78.18 120.16 23.6228.4 Stage 1 crystal wash 218 24.3 52.97 44.19 23.4 Stage 1 washedcrystals 54.4 78.38 42.64 4.62 2.0 to Still 789 42.68 336.76 64.04 215.72nd crystal feed 444.3 67.70 300.79 66.81 201.0 Still wash 196 1.2 2.3550.01 1.2 Stage 2 liquor 315 63.5 200.03 85.57 171.2 Stage 2 crystals165.25 82.19 135.82 37.30 50.7 Stage 2 crystal wash 248 27 66.96 60.3740.4 Stage 2 washed crystals 77.65 77.52 60.19 9.33 5.6 Pasteurizedproduct 314.3 63.01 198.05 86.00 170.3The composition of the product (pasteurized product) so made is shown inFIG. 39.

The composition described herein is discrete, and can be differentiatedvia chromatography, from the major commercial IMO products that areavailable today. A comparison of the ISOThrive™ composition, which isheretofore canon, with the commercial prebiotic IMO-based compositions,is given in FIG. 35.

A summary of the process illustrated in Example 7 is shown in FIG. 36.

Example 8

Four more batches were produced in 3000 liter batches using the methodsdescribed in the foregoing Examples. These four batches had batchnumbers 150622, 151105, 160120, and 161202, and had the followingcompositions.

Batch 150622 151105 160120 161202 brix 67.35 69.33 69.69 64.57 glycerol0.55 0.39 0.46 0.34 erythritol 0.27 0.15 0.18 0.16 mannitol 8.01 6.235.97 5.96 glucose 0.42 0.92 1.10 0.92 fructose 0.05 0.15 0.16 0.13leucrose 2.13 2.03 1.79 1.81 sucrose 0.62 0.69 0.65 0.79 maltose 2.983.85 3.73 4.34 1,6-DP2 0.21 0.14 0.13 0.27 1,4-DP3 1.65 1.82 1.84 1.931,6-DP3 0.28 0.43 0.44 0.47 1,6-DP4 0.32 0.53 0.56 0.54 MIMO-DP3 9.9411.04 10.44 12.27 MIMO-DP4 22.76 24.13 23.49 25.53 MIMO-DP5 26.27 26.4726.56 26.32 MIMO-DP6 15.74 14.54 15.32 13.16 MIMO-DP7 5.09 4.34 4.723.53 MIMO-DP8 2.06 1.60 1.89 1.20 MIMO-DP9 0.65 0.54 0.56 0.32 lacticacid 0.00 0.00 0.01 0.02 acetic acid 0.00 0.00 0.00 0.00 Purity, % 83.1183.63 83.98 83.34 Mw, Da 782.7 763.9 773.6 745.5

Example 9

This Example shows that media from growth of Lactobacillus gasseri orLactococcus lactis can affect the morphology and growth of head and necksquamous cell carcinoma (HNSCC) cells.

Methods

Overnight cultured head and neck squamous cell carcinoma (HNSCC) cells(HSC-3 and 14A cells; 70-80% confluency) were treated with differentconcentrations (25, 50, 100, 150, 200 and 300 mg/ml; BCA protein assay)of broth obtained from Lactobacillus gasseri ATCC 4962 or Lactococcuslactis subsp. lactis NRRL B-1821 bacteria for 2 hours. After 2 hours themedia was removed and replaced with regular medium and the carcinomacells were then cultured for another 12 hours. No bacterialcontamination or precipitation of the cancer cell media was observedduring the incubation.

Results

Cell growth inhibition and morphological changes were induced byLactobacillus gasseri and Lactococcus lactis in HSC-3 cells in adose-dependent manner. Slight induction of morphological changes in thesquamous cell carcinoma (HNSCC; 14A) cells were also observed. Thesepreliminary results indicate that broth from Lactobacillus gasseri maybe a more effective inhibitor of head and neck squamous cell carcinomacell growth than broth from Lactococcus lactis.

These results also show that cancer cells can be treated withconditioned media continuously for 12-18 hours (overnight).

Example 10

This Example illustrates that culture medium obtained after growth ofLactobacillus gasseri ATCC 4962 or Lactococcus lactis subsp. lactis NRRLB-1821 can inhibit colon cancer cell growth.

Cell Proliferation Assay

Growth of HCT-15 and DLD-1 colorectal cancer cells was examined duringexposure to media obtained after growth of Lactobacillus gasseri ATCC4962 or Lactococcus lactis subsp. lactis NRRL B-1821. The HCT-15 cellsare human colonic epithelium, adherent, Dukes' type C colorectal cancercells. The DLD-1 cells are human colonic epithelium, adherent, Dukes'type C colorectal cancer cells differentiated from HCT-15 andoriginating from different chromosomal aberrations (but the mutationsare within the HCT-15 parent cell line).

HCT-15 and DLD-1 colorectal cancer cells were incubated for 24 hourswith control media or cell-free broth from Lactobacillus gasseri ATCC4962 or Lactococcus lactis subsp. lactis NRRL B-1821 cultures (100, 200,400 or 800 μg/mL).

To determine the effect of broth from Lactobacillus gasseri ATCC 4962 orLactococcus lactis subsp. lactis NRRL B-1821 cultures on cancer cellproliferation, a CyQUANT NF Cell Proliferation Assay Kit was usedaccording to manufacturer's instructions (Invitrogen/Life Technologies,Grand Island, N.Y.).

Results

As illustrated in FIG. 37A-37B, broth from Lactobacillus gasseri andLactococcus lactis cultures reduced colon cancer cell proliferation in adose-dependent manner relative to control culture media. These dataindicate that Lactobacillus gasseri and Lactococcus lactis secrete asubstance that inhibits cancer cell growth.

Example 11

This Example illustrates that Weissella viridescens growth is inhibitedby nisins A and Z. Bacteria assigned to the genus Weissella areGram-positive, catalase-negative, non-endospore forming cells withcoccoid or rod-shaped morphology. Weissella viridescens can be anopportunistic pathogen involved in human infections.

Materials and Methods

Purified nisin A and Z are commercially available and were acquired fromHandary S. A. (Brussels Belgium). Certificates of analysis for each lotindicated purities of 95.2 and 99.6% for nisin A and Z, respectively.

100 g De-Man, Rogosa and Sharpe (MRS) media was prepared in a 500 mLErlenmeyer to contain 5.5% solids in deionized water (18.2 MΩ, HydroService and Supplies, Gaithersburg, Md.). The media was autoclaved (121°C. 15 min), cooled, and inoculated with 1 mL (0.5 mL culture+0.5 mLglycerol, 40%; frozen at −78° C.) of Weissella viridescens NRRL B-1951(test strain). The culture was incubated at 31° C. overnight (15 Hr).

The next day, stock solutions of either nisin A or Z were prepared indeionized water to contain 31.3 and 33.7 μg/g of each peptide.

100 g MRS media was prepared to contain 5.5% solids. Two sets (one seteach for nisin A and nisin Z) of eight Hach-type tubes (Kimble Chase45066-16100) were prepared. Within each set, in order was added 5.00,4.95, 4.90, 4.75, 4.50, 4.00, 3.50 and 3.00 g MRS media. The tubes weresealed and autoclaved.

To each tube was added, of the appropriate nisin stock, 0.00, 0.05,0.10, 0.25, 0.50, 1.00, 1.50, and 2.00 g. Each tube was inoculated with0.25 g of late-log W. viridescens NRRL B-1951 culture. The tubes weresealed and the absorbance at 600 nm was quickly measured (Hach DR900).The tubes were segregated by set and incubated at 31° C. overnight (15Hr).

Each tube was sampled (1.5 mL conical polypropylene centrifuge tubes)and centrifuged at 10 kRPM for 10 minutes to remove and suspended cells.The resulting supernatants were filtered (0.2 μm nylon), diluted to 0.5%solids, and analyzed by high pressure liquid chromatography (HPLC,Agilent 1100, refractive index detector and BioRad Ainex HPX-87H column)for consumption of glucose and production of organic acids. Thefollowing table illustrates the concentration of sugars such as glucoseand production of organic acids during the incubation.

Concentration of Sugars and Organic Acids in the Fermentation Media%/brix A1 A2 A4 A5 A6 A7 A8 Maltotriose 0.401 0.312 0.298 0.276 0.2880.419 0.305 maltose 1.078 0.581 0.815 0.598 0.941 0.775 0.739 glucose8.883 23.122 24.138 24.698 25.304 25.444 25.537 fructose 1.122 0.0000.000 0.000 0.000 0.000 0.000 lactic acid 18.404 6.884 5.496 4.672 2.7071.521 1.579 glycerol 0.386 0.292 0.319 0.350 0.328 0.299 0.301 formicacid 0.302 0.263 0.282 0.407 0.280 0.234 0.233 acetic acid 10.267 7.9678.219 8.296 8.143 7.899 8.091 ethanol 3.979 0.068 0.062 0.094 0.0570.055 0.071 %/brix Z1 Z2 Z4 Z5 Z6 Z7 Z8 Maltotriose 0.406 0.282 0.2650.311 0.247 0.291 0.318 maltose 0.810 0.722 1.007 0.750 0.726 0.8950.876 glucose 10.064 22.988 24.153 24.698 25.353 25.501 25.740 fructose0.000 0.000 0.000 0.000 0.000 0.000 0.000 lactic acid 17.820 6.742 5.0812.995 1.351 1.431 1.652 glycerol 0.404 0.343 0.337 0.321 0.320 0.3350.346 formic acid 0.331 0.303 0.309 0.281 0.267 0.263 0.299 acetic acid10.399 8.273 8.247 8.026 7.969 8.026 8.261 ethanol 3.783 0.057 0.0830.067 0.059 0.065 0.148

Note that the MRS media originally contained 7% acetate; therefore 7%should be subtracted from the values for acetate in the above table toascertain the amount of acetate generated during the assay.

Each tube was thoroughly mixed to suspend settled cells and theabsorbance was measured again at 600 nm. The final absorbance values foreach sample were corrected for their respective background (taken justafter inoculation, scattered light from added cells). The correctedabsorption values were a measure of W. viridescens growth. The correctedvalues were plotted against nisin concentration and minimum inhibitingconcentration approximated via regression. Exemplary absorbance resultsare provided in the table below.

W. viridescens Culture Absorbance (600 nm) Tube MRS, Test Nisin, 9141691516 Corrected #: g: med., g: inoc, g: gtot: μg/g: ABS 610i: ABS 610f:ABS 610rs: A1 5.05046 0.00000 0.24848 5.29894 0.00 0.465 2.333 1.868 A24.96543 0.05497 0.24044 5.26084 0.33 0.462 1.568 1.106 A3 4.883220.11358 0.25644 5.25324 0.68 0.468 1.531 1.063 A4 4.75224 0.231200.26348 5.24692 1.38 0.466 1.334 0.868 A5 4.52853 0.52264 0.2531 5.304373.08 0.438 0.911 0.473 A6 4.01513 1.00698 0.24562 5.26773 5.97 0.420.536 0.116 A7 3.50321 1.4864 0.24991 5.23952 8.86 0.402 0.403 0.001 A83.01078 1.99641 0.24476 5.25195 11.87 0.36 0.361 0.001 Z1 5.035940.00000 0.25228 5.28822 0.00 0.469 2.361 1.892 Z2 4.94155 0.053450.25405 5.24905 0.34 0.472 1.618 1.146 Z3 4.89029 0.12073 0.254785.26580 0.77 0.464 1.526 1.062 Z4 4.75115 0.24831 0.25259 5.25205 1.580.455 1.296 0.841 Z5 4.52267 0.50221 0.25131 5.27619 3.19 0.442 0.8010.359 Z6 4.03195 0.99365 0.25415 5.27975 6.31 0.423 0.426 0.003 Z73.50064 1.49004 0.24365 5.23433 9.54 0.383 0.384 0.001 Z8 3.061592.00403 0.25834 5.32396 12.61 0.385 0.386 0.001The data shown in the above table are plotted in FIG. 38, whichgraphically illustrates that minimal inhibitory concentration of eachpeptide was between 5 and 7 μg/g.

These data indicate that nisin A and nisin Z can effectively inhibitbacterial (Weissella viridescens NRRL B-1951) growth.

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Factors Affecting a-1,2    Glucooligosaccharide Synthesis by Leuconostoc mesenteroides NRRL    B-1299 Dextransucrase. Biotechnol. Bioeng. 74 (6), pp. 498-504.-   19. Chludzinski, A. M., Germaine, G. R. and Schachtele, C. F.    (1974). Purification and Properties of Dextransucrase from    Streptococcus mutans. J. Bacteriol. 118 (1), pp. 1-7.-   20. Miller, A. W., Eklund, S. H. and Robyt, J. F. (1986). Milligram    to gram scale purification and characterization of dextransucrase    from Leuconostoc mesenteroides NRRL B-512F. Carbohydr. Res. 147 (1),    pp. 119-133.-   21. Goyal, A., Nigam, M., and Katiyar, S. S. (1995). Optimal    conditions for production of dextransucrase from Lecuonostoc    mesenteroides NRLL (NRRL) B-512F and its properties. J. Basic    Microbiol. 35 (6), pp. 375-384.-   22. Sarwat, F., Ul Qader, S-A., Aman, A. and Ahmed, N. (2008).    Production & Characterization of a Unique Dextran from an Indigenous    Leuconostoc mesenteroides CMG713. Int. J. Biol. 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All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby specifically incorporated by reference to the same extent asif it had been incorporated by reference in its entirety individually orset forth herein in its entirety. Applicants reserve the right tophysically incorporate into this specification any and all materials andinformation from any such cited patents or publications.

The following statements describe and summarize various embodiments ofthe invention according to the foregoing description in thespecification.

Statements:

-   -   1. A composition comprising maltosyl-isomaltooligosaccharides        with a mass average molecular weight distribution of about 640        to 1000 daltons.    -   2. The composition of statement 1, comprising a mass average        molecular weight distribution of about 730 to 900 daltons.    -   3. The composition of statement 1 or 2, where the        maltosyl-isomaltooligosaccharides contain more α-(1→6) glucosyl        linkages than α-(1,2), α-(1,3), or α-(1,4) glucosyl linkages.    -   4. The composition of any of statements 1-3, where at least 60%,        or at least 70%, or at least 75%, or at least 80%, or at least        85%, or at least 90% of the maltosyl-isomaltooligosaccharides        have at least 50% α-(1,6) glucosyl linkages, or at least 52%        α-(1,6) glucosyl linkages, or at least 55% α-(1,6) glucosyl        linkages, or at least 60% α-(1,6) glucosyl linkages, or at least        65% α-(1,6) glucosyl linkages, or at least 70% α-(1,6) glucosyl        linkages, or at least 75% α-(1,6) glucosyl linkages, or at least        80% α-(1,6) glucosyl linkages, or at least 85% α-(1,6) glucosyl        linkages, or at least 87% α-(1,6) glucosyl linkages, or at least        89% α-(1,6) glucosyl linkages, or at least 90% α-(1,6) glucosyl        linkages.    -   5. The composition of any of statements 1-3, where the        maltosyl-isomaltooligosaccharides have at least 80%, or at least        85%, or at least 89% α-(1,6) glucosyl linkages, or at least 90%        α-(1,6) glucosyl linkages as detected by HPLC or NMR.    -   6. The composition of any of statements 1-5, where the        maltosyl-isomaltooligosaccharides can optionally have one or two        α-(1,4) linkages.    -   7. The composition of any of statements 1-6, where the        maltosyl-isomaltooligosaccharides can optionally have one        [—O-α-(1,4)-] linkage at the reducing end.    -   8. The composition of any of statements 1-7, where the        maltosyl-isomaltooligosaccharides have no more than about 18        glucose units, or no more than about 16 glucose units, or no        more than about 15 glucose units, or no more than about 14        glucose units, or no more than about 13 glucose units, or no        more than about 12 glucose units, or no more than about 11        glucose units, or no more than about 10 glucose units as        detected by HPAEC-PAD or HPLC-RID.    -   9. The composition of any of statements 1-8, where the        maltosyl-isomaltooligosaccharides have a maltose unit at the        reducing end.    -   10. The composition of any of statements 1-9, with less than        2%/brix isomaltose, or less than 1%/brix isomaltose, or less        than 0.5%/brix isomaltose, or less than 0.2%/brix isomaltose, or        less than 0.1%/brix isomaltose as detected by HPAEC-PAD or        HPLC-RID.    -   11. The composition of any of statements 1-10, with less than        5%/brix glucose, or less than 4%/brix glucose, or less than        3%/brix glucose, or less than 2%/brix glucose, or less than        1%/brix glucose as detected by HPAEC-PAD or HPLC-RID.    -   12. The composition of any of statements 1-11, with less than        5%/brix sucrose, or less than 4%/brix sucrose, or less than        3%/brix sucrose, or less than 2%/brix sucrose, or less than        1%/brix sucrose as detected by HPAEC-PAD or HPLC-RID.    -   13. The composition of any of statements 1-12, with less than        4%/brix fructose, or less than 3%/brix fructose, or less than        2%/brix fructose, or less than 1%/brix fructose, or less than        0.5%/brix fructose, or less than 0.25%/brix fructose as detected        by HPAEC-PAD or HPLC-RID.    -   14. The composition of any of statements 1-13, with less than        7%/brix lactate, or less than 6%/brix lactate, or less than        5%/brix lactate, or less than 3%/brix lactate, or less than        2%/brix lactate, or less than 1%/brix lactate, or less than        0.5%/brix lactate, or less than 0.2%/brix lactate, or less than        0.1%/brix lactate as detected by HPAEC-PAD or HPLC-RID.    -   15. The composition of any of statements 1-14, with less than        8%/brix maltose, or less than 7%/brix maltose, or less than        6%/brix maltose, or less than 5%/brix maltose as detected by        HPAEC-PAD or HPLC-RID.    -   16. The composition of any of statements 1-15, with more than        3%/brix mannitol, or more than 4%/brix mannitol, or more than        5%/brix mannitol as detected by HPAEC-PAD or HPLC-RID.    -   17. The composition of any of statements 1-16, with less than        30%/brix mannitol, or less than 20%/brix mannitol, or less than        15% mannitol or less than 12%/brix mannitol, or less than 10%        mannitol, or less than 9%/brix mannitol, or less than 8%        mannitol as detected by HPAEC-PAD or HPLC-RID.    -   18. The composition of any of statements 1-17, with the        compositions less than 4%/brix glycerol, or less than 3%/brix        glycerol, or less than 2%/brix glycerol, or less than 1%/brix        glycerol, or less than 0.6%/brix glycerol, or less than        0.5%/brix glycerol detectable by HPLC-RID or HPLC-RID.    -   19. The composition of any of statements 1-18, with less than        20%/brix MIMO-DP3, or less than 19%/brix MIMO-DP3, or less than        18%/brix MIMO-DP3, or less than 17%/brix MIMO-DP3, or less than        16%/brix MIMO-DP3, or less than 15%/brix MIMO-DP3.    -   20. The composition of any of statements 1-19, with less than        30%/brix MIMO-DP4, or less than 28%/brix MIMO-DP4, or less than        27%/brix MIMO-DP4, or less than 26%/brix MIMO-DP4, or less than        25%/brix MIMO-DP4, or less than 24%/brix MIMO-DP4, or less than        23%/brix MIMO-DP4.    -   21. The composition of any of statements 1-20, with more than        18%/brix MIMO-DP5, or more than 19%/brix MIMO-DP5, or more than        20%/brix MIMO-DP5, or more than 21%/brix MIMO-DP5, or more than        22%/brix MIMO-DP5, or more than 23%/brix MIMO-DP5, or more than        23.5%/brix MIMO-DP5, or more than 24%/brix MIMO-DP5, or more        than 25%/brix MIMO-DP5.    -   22. The composition of any of statements 1-21, with more than        10%/brix MIMO-DP6, or more than 11%/brix MIMO-DP6, or more than        12%/brix MIMO-DP6, or more than 13%/brix MIMO-DP6, or more than        14%/brix MIMO-DP6, or more than 14.5%/brix MIMO-DP6, or more        than 15%/brix MIMO-DP6.    -   23. The composition of any of statements 1-22, with more than        1%/brix MIMO-DP7, or more than 2%/brix MIMO-DP7, or more than        3%/brix MIMO-DP7, or more than 3.5%/brix MIMO-DP7, or more than        4%/brix MIMO-DP7, or more than 5%/brix MIMO-DP7, or more than        5.5%/brix MIMO-DP7.    -   24. The composition of any of statements 1-23, with more than        0.5%/brix MIMO-DP8, or more than 1%/brix MIMO-DP8, or more than        1.5%/brix MIMO-DP8, or more than 1.75%/brix MIMO-DP8, or more        than 2%/brix MIMO-DP8, or more than 2.25%/brix MIMO-DP8.    -   25. The composition of any of statements 1-24, with more than        0.1%/brix MIMO-DP9, or more than 0.2%/brix MIMO-DP9, or more        than 0.3%/brix MIMO-DP9, or more than 0.4%/brix MIMO-DP9, or        more than 0.5%/brix MIMO-DP9.    -   26. The composition of any of statements 1-25, containing the        components shown in FIG. 39.    -   27. The composition of any of statements 1-26, as a concentrated        solution.    -   28. The composition of any of statements 1-27, dried as a        powder, for example, by drying, spray drying or by        freeze-drying.    -   29. The composition of any of statements 1-28, aliquoted into        individual servings for human or animal consumption.    -   30. The composition of any of statements 1-29, aliquoted into        individual serving of about 0.25 ml to about 10 ml, or about 0.5        ml to about 8 ml, or about 0.75 ml to about 7 ml, or about 1 ml        to about 5 ml, or about 1 ml to about 3 ml.    -   31. A method comprising administering the composition of any of        statements 1-30 to an animal (e.g., a human or a domesticated        animal).    -   32. A method for the preparation of a composition comprising:        -   (a) contacting a feedstock comprising Leuconostoc citreum            ATCC 13146 (NRRL B-742) bacterial cells with a ratio of            sucrose to maltose ranging from 2.0 to about 4.5 in an            aqueous culture to form a fermentation mixture;        -   (b) fermenting the fermentation mixture at a pH between 4            and 8;        -   (c) removing the bacterial cells to generate a cell-free            liquor;        -   (d) polishing the cell-free liquor by removal of insoluble            impurities; decolorization (e.g., using activated charcoal,            activated carbon, a weak base anion resin, or a combination            thereof), de-aching (e.g., using a strong acid cation resin            to remove metal ions, or using a two-step process using a            strong acid followed by a weak base); removing protein            (e.g., by heating, evaporating the aqueous culture medium,            and centrifugation or filtration, or by using a weak base            anion resin); removing organic acids (e.g., utilizing a weak            base anion resin, liquid chromatography using a            chromatographic grade gel-type strong acid cation exchange            resin in calcium form (SAC-Ca⁺⁺); or any combination            thereof, to generate a polished product;        -   e) washing insoluble impurities, decolorization agents,            de-ashing agents, evaporating mechanisms (e.g., a wiped film            evaporator), centrifugation pellets, filters, resins, weak            base anion resins, chromatographic resins, chromatographic            grade gel-type or macroporous strong acid cation exchange            resins, or any combination thereof to generate one or more            washes; and combining one or more washes together, or with            the cell-free liquor; or with the polished product;        -   wherein the final composition comprises            maltosyl-isomaltooligosaccharides with a mass average            molecular weight distribution of about 640 to 1000 daltons            and at least 3% mannitol.    -   33. The method of statement 32, where the        maltosyl-isomaltooligosaccharides have at least 50% α-(1,6)        glucosyl linkages, or at least 52% α-(1,6) glucosyl linkages, or        at least 55% α-(1,6) glucosyl linkages, or at least 60% α-(1,6)        glucosyl linkages, or at least 65% α-(1,6) glucosyl linkages, or        at least 70% α-(1,6) glucosyl linkages, or at least 75% α-(1,6)        glucosyl linkages, or at least 80% α-(1,6) glucosyl linkages, or        at least 85% α-(1,6) glucosyl linkages, or at least 89% α-(1,6)        glucosyl linkages, or at least 90% α-(1,6) glucosyl linkages.    -   34. The method of statement 32 or 33, where the ratio of sucrose        to maltose ranges from about 2.0 to about 4.5, from about 2.2 to        about 4.3, or about 2.3 to about 4.0, or about 2.4 to about 4.0,        or about 2.5 to about 3.75, or about 2.5 to about 3.5, or about        2.5 to about 3.0, or about 2.75.    -   35. A composition generated by a method comprising        -   (a) contacting a feedstock comprising Leuconostoc citreum            ATCC 13146 (NRRL B-742) bacterial cells with a ratio of            sucrose to maltose ranging from 2.0 to about 4.5 in an            aqueous culture to form a fermentation mixture;        -   (b) fermenting the fermentation mixture at a pH between 4            and 8;        -   (c) removing the bacterial cells to generate a cell-free            liquor;        -   (d) polishing the cell-free liquor by removal of insoluble            impurities; decolorization (e.g., using activated charcoal,            activated carbon, a weak base anion resin, or a combination            thereof), de-aching (e.g., using a strong acid cation resin            to remove metal ions, or using a two-step process using a            strong acid followed by a weak base); removing protein            (e.g., by heating, evaporating the aqueous culture medium,            and centrifugation or filtration, or by using a weak base            anion resin); removing organic acids (e.g., utilizing a weak            base anion resin, liquid chromatography using a            chromatographic grade gel-type strong acid cation exchange            resin in calcium form (SAC-Ca⁺⁺); or any combination            thereof, to generate a polished product;        -   e) washing insoluble impurities, decolorization agents,            de-ashing agents, evaporating mechanisms (e.g., a wiped film            evaporator), centrifugation pellets, filters, ration, weak            base anion resins, chromatographic resins, chromatographic            grade gel-type strong acid cation exchange resins, or any            combination thereof to generate one or more washes; and            combining one or more washes together, or with the cell-free            liquor; or with the polished product;        -   wherein the final composition comprises            maltosyl-isomaltooligosaccharides with a mass average            molecular weight distribution of about 640 to 1000 daltons            and at least 3% mannitol.    -   36. The composition of statement 35, where at least 60%, or at        least 70%, or at least 75%, or at least 80%, or at least 85%, or        at least 90% of the maltosyl-isomaltooligosaccharides have at        least 50% α-(1,6) glucosyl linkages, or at least 52% α-(1,6)        glucosyl linkages, or at least 55% α-(1,6) glucosyl linkages, or        at least 60% α-(1,6) glucosyl linkages, or at least 65% α-(1,6)        glucosyl linkages, or at least 70% α-(1,6) glucosyl linkages, or        at least 89% α-(1,6) glucosyl linkages, or at least 90% α-(1,6)        glucosyl linkages.    -   37. The composition of statement 35 or 36, where the sucrose to        maltose ranges from about 2.0 to about 4.5, from about 2.2 to        about 4.3, or about 2.3 to about 4.0, or about 2.4 to about 4.0,        or about 2.5 to about 3.75, or about 2.5 to about 3.5, or about        2.5 to about 3.0, or about 2.75.

The specific compositions and methods described herein arerepresentative, exemplary and not intended as limitations on the scopeof the invention. Other objects, aspects, and embodiments will occur tothose skilled in the art upon consideration of this specification, andare encompassed within the spirit of the invention as defined by thescope of the claims. It will be readily apparent to one skilled in theart that varying substitutions and modifications can be made to theinvention disclosed herein without departing from the scope and spiritof the invention. The terms and expressions that have been employed areused as terms of description and not of limitation, and there is nointent in the use of such terms and expressions to exclude anyequivalent of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention as claimed. Thus, it will be understood thatalthough the present invention has been specifically disclosed byembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims andstatements of the invention.

The invention illustratively described herein may be practiced in theabsence of any element or elements, or limitation or limitations, whichis not specifically disclosed herein as essential. The methods andprocesses illustratively described herein may be practiced in differingorders of steps, and the methods and processes are not necessarilyrestricted to the orders of steps indicated herein or in the claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a compound” or “anoligosaccharide” or “a maltose” includes a plurality of such compounds,oligosaccharides, or maltose sugars, and so forth. In this document, theterm “or” is used to refer to a nonexclusive or, such that “A or B”includes “A but not B,” “B but not A,” and “A and B,” unless otherwiseindicated.

Under no circumstances may the patent be interpreted to be limited tothe specific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

The Abstract is provided to comply with 37 C.F.R. § 1.72(b) to allow thereader to quickly ascertain the nature and gist of the technicaldisclosure. The Abstract is submitted with the understanding that itwill not be used to interpret or limit the scope or meaning of theclaims.

What is claimed is:
 1. A composition comprisingmaltosyl-isomaltooligosaccharides with a mass average molecular weightdistribution of about 730 to 900 daltons, and where at least 60% of themaltosyl-isomaltooligosaccharides have at least 75% α-(1,6) linkages,and wherein at least 40% of the maltosyl-isomaltooligosaccharides in thecomposition have a degree of polymerization (DP) of 5 or more.
 2. Thecomposition of claim 1, where the maltosyl-isomaltooligosaccharides haveno more than about 18 glucose units.
 3. The composition of claim 1,where the maltosyl-isomaltooligosaccharides have a maltose unit at thereducing end.
 4. The composition of claim 1, with less than 2%/brixisomaltose.
 5. The composition of claim 1, with less than 5%/brixglucose.
 6. The composition of claim 1, with less than 5%/brix sucrose.7. The composition of claim 1, with less than 4%/brix fructose.
 8. Thecomposition of claim 1, with less than 7%/brix lactate.
 9. Thecomposition of claim 1, with less than 8%/brix maltose.
 10. Thecomposition of claim 1, with 3% or more than 3%/brix mannitol.
 11. Thecomposition of claim 1, with less than 4%/brix glycerol.
 12. Thecomposition of claim 1, with less than 20%/brix MIMO-DP3.
 13. Thecomposition of claim 1, with less than 30%/brix MIMO-DP4.
 14. Thecomposition of claim 1, with more than 18%/brix MIMO-DP5.
 15. Thecomposition of claim 1, with more than 10%/brix MIMO-DP6.
 16. Thecomposition of claim 1, with more than 1%/brix MIMO-7.
 17. Thecomposition of claim 1, with more than 0.5%/brix MIMO-DP8.
 18. Thecomposition of claim 1, with more than 0.1%/brix MIMO-DP9.
 19. Thecomposition of claim 1, as a concentrated solution.
 20. The compositionof claim 1, dried as a powder.
 21. The composition of claim 1, aliquotedinto individual servings for human or animal consumption.
 22. Thecomposition of claim 1, aliquoted into individual serving of about 0.25ml to about 10 ml.
 23. A composition generated by a method comprising(a) contacting a feedstock comprising Leuconostoc citreum ATCC 13146(NRRL B-742) bacterial cells with a fixed ratio of sucrose to maltoseranging from 2.1 to about 4.5 in an aqueous culture to form afermentation mixture; (b) fermenting the fermentation mixture at a pHbetween 4 and 8; (c) removing the bacterial cells to generate acell-free liquor; (d) polishing the cell-free liquor by removal ofinsoluble impurities; decolorization; de-ashing); removing protein;removing organic acids; or any combination thereof, to generate apolished product; e) washing insoluble impurities, decolorizationagents, de-ashing agents, evaporating mechanisms, centrifugationpellets, filters, resins, weak base anion resins, chromatographicresins, chromatographic grade gel-type strong acid cation exchangeresins, or any combination thereof to generate one or more washes; andcombining the one or more washes together, or combining the one or morewashes with the cell-free liquor; or combining the one or more washeswith the polished product; wherein the final composition comprisesmaltosyl-isomaltooligosaccharides with a mass average molecular weightdistribution of about 730 to 900 to daltons, where at least 60% of themaltosyl-isomaltooligosaccharides have at least 75% α-(1,6) linkages,and wherein at least 40% of the maltosyl-isomaltooligosaccharides in thecomposition have a degree of polymerization (DP) of 5 or more.